#### *Control of the S6Ks by mTOR*

Another important target of mTORC1 is the S6 kinases family, including S6K1 and S6K2. Ribosomal protein S6 (rpS6) is highly phosphorylated protein containing at least five phosporylating sites in its C-terminus. There are two main classes of protein kinases which are responsible for rpS6 phosphorylation *in vitro*, namely the p70 S6 kinases (S6Ks) and the p90 ribosomal protein S6 kinases, also known as RSKs [164, 165], (reviewed in [151]). The observed sensitivity of rpS6 to rapamycin lead to the speculation that S6K are responsible for rpS6 phosphorylation as their activation is mediated by mTOR. Unlike S6Ks, the RSKs are not influenced by rapamycin since they are known to be activated through the classical MAP kinase (ERK) pathway (see below). There are two similar S6 kinase proteins, S6K1 and S6K2, in mammals [166], which show 70% of amino acid homology. Each p70S6K gene encodes two distinct proteins due to alternative splicing of the mRNAs. Several data confirm that activation of both the S6K1 and S6K2 are regulated by mTORC1 [118, 167, 168]. S6K1, which was discovered earlier than S6K2, is ubiquitously expressed and appears to be more critical for the control of cell growth. S6K1 can be activated by a wide variety of extracellular signals and is known as the major rpS6 kinase in mammalian cells and key player in the control of cell growth (cell size) and proliferation [169, 170].

Earlier it was thought that activated S6K1 regulates translation of a class of mRNA transcripts that bear a 5'-terminal oligopolypyrimidine (5'-TOP). Particularly, it was shown that S6K1 phosphorylates eIF4B on S422, which is located in the RNA binding region that is necessary for promoting the helicase activity of eIF4A [171]. Few data indicate that eIF4B phosphorylation by S6K1 is both sufficient and necessary for its recruitment to the translation preinitiation complex [172]. However, there are also some data that disprove this model. In S6K1/2–/– cells, 5'-TOP mRNA translation is intact and still rapamycin-sensitive [173]. These results are in concordance with earlier data showing that mitogenic-stimulated or amino acid dependent 5'-TOP mRNAs translation is dependent on PI3K mediated signaling, and does not require S6K1 activity and ribosomal protein S6 phosphorylation [174, 175]. Instead, a role for the S6 kinases in controlling the cell size has been suggested as deletion of S6K leads to animal size decrease [173]. Studies performed on 'knock-in' mice in which all sites phosphorylated by the S6 kinases were mutated also indicated a role for S6 phosphorylation in cell growth [176]. These knock-in cells still demonstrated faster rates of protein synthesis at the same time being decreased in size. This could be explained by elevated access of the S6K to other substrates involved in translation, such as eIF4B and eEF2 kinase (see below) [151].

Another pool of data connecting S6K1 activity and translation initiation occurs from the study of potential tumor suppressor, Programmed cell death 4 (PDCD4) protein **(Figure**  **5A).** PDCD4 binds to eIF4A and is thought to inhibit its helicase activity [177]. PDCD4 is also thought to prevent eIF4A from incorporating into the eIF4F complex by competing with eIF4G for eIF4A binding [178]. S6K1 phosphorylates PDCD4 on S67 in response to growth factor stimulation resulting in its subsequent degradation through the ubiquitin ligase βTrCP101. Therefore, S6K1-dependent phosphorylation of PDCD4 prevents the inhibitory effect of PDCD4 towards eIF4A helicase function within the eIF4F complex.

22 Protein Phosphorylation in Human Health

*Control of the S6Ks by mTOR*

eEF2 kinase (see below) [151].

data evidence that phosphorylation is mediated by mTORC1: (i) it is inhibited by starvation of cells for amino acids; (ii) it is activated by Rheb; (iii) it is suppressed by TSC1/2; (iv) it is sensitive to inhibitors of the kinase activity of mTOR and (v) it is decreased in cells in which mTOR levels have been knocked down [158]. Therefore, further study of this process needs

Another important target of mTORC1 is the S6 kinases family, including S6K1 and S6K2. Ribosomal protein S6 (rpS6) is highly phosphorylated protein containing at least five phosporylating sites in its C-terminus. There are two main classes of protein kinases which are responsible for rpS6 phosphorylation *in vitro*, namely the p70 S6 kinases (S6Ks) and the p90 ribosomal protein S6 kinases, also known as RSKs [164, 165], (reviewed in [151]). The observed sensitivity of rpS6 to rapamycin lead to the speculation that S6K are responsible for rpS6 phosphorylation as their activation is mediated by mTOR. Unlike S6Ks, the RSKs are not influenced by rapamycin since they are known to be activated through the classical MAP kinase (ERK) pathway (see below). There are two similar S6 kinase proteins, S6K1 and S6K2, in mammals [166], which show 70% of amino acid homology. Each p70S6K gene encodes two distinct proteins due to alternative splicing of the mRNAs. Several data confirm that activation of both the S6K1 and S6K2 are regulated by mTORC1 [118, 167, 168]. S6K1, which was discovered earlier than S6K2, is ubiquitously expressed and appears to be more critical for the control of cell growth. S6K1 can be activated by a wide variety of extracellular signals and is known as the major rpS6 kinase in mammalian cells and key

Earlier it was thought that activated S6K1 regulates translation of a class of mRNA transcripts that bear a 5'-terminal oligopolypyrimidine (5'-TOP). Particularly, it was shown that S6K1 phosphorylates eIF4B on S422, which is located in the RNA binding region that is necessary for promoting the helicase activity of eIF4A [171]. Few data indicate that eIF4B phosphorylation by S6K1 is both sufficient and necessary for its recruitment to the translation preinitiation complex [172]. However, there are also some data that disprove this model. In S6K1/2–/– cells, 5'-TOP mRNA translation is intact and still rapamycin-sensitive [173]. These results are in concordance with earlier data showing that mitogenic-stimulated or amino acid dependent 5'-TOP mRNAs translation is dependent on PI3K mediated signaling, and does not require S6K1 activity and ribosomal protein S6 phosphorylation [174, 175]. Instead, a role for the S6 kinases in controlling the cell size has been suggested as deletion of S6K leads to animal size decrease [173]. Studies performed on 'knock-in' mice in which all sites phosphorylated by the S6 kinases were mutated also indicated a role for S6 phosphorylation in cell growth [176]. These knock-in cells still demonstrated faster rates of protein synthesis at the same time being decreased in size. This could be explained by elevated access of the S6K to other substrates involved in translation, such as eIF4B and

Another pool of data connecting S6K1 activity and translation initiation occurs from the study of potential tumor suppressor, Programmed cell death 4 (PDCD4) protein **(Figure**

to clarify the molecular mechanisms of mTOR downstream signaling.

player in the control of cell growth (cell size) and proliferation [169, 170].

Recent data give new evidence on interconnection of the mTOR/S6K1 pathway and translation preinitiation complex assembly [172, 179]. Under the poor growth conditions, S6K1 but not mTORC1 binds with multisubunit initiation factor eIF3 that was identified as a dynamic scaffold for mTORC1 and S6K1 binding [172] **(Figure 5B).** Upon growth factors or nutrients availability, the mTORC1 is recruited to eIF3 and phosphorylates S6K1. Based on polysome analysis and cap-binding assays, it is thought that the mTORC1–eIF3 complex associates with the mRNA 5′ cap, bringing mTORC1 into proximity with 4EBP1. Phosphorylation of S6K1 at T389 leads to its dissociation from eIF3. T389-phosphorylated S6K1 binds to PDK1 **(Figure 5A**), which phosphorylates S6K1 at T229. The fully activated S6K1 is able to phosphorylate eIF4B and S6. Phosphorylation of eIF4B by S6K1 at S422 promotes its association with eIF3 [172, 180]. The interaction of mTOR with eIF3 also strengthens the association between eIF4G and eIF3 [181]. Described interactions cooperate to enhance the assembly of translation initiation complex and facilitate cap-dependent translation.

The S6 kinases are activated by phosphorylation at multiple sites. Several of them lie within the C-terminus, while two others lay immediately C-terminal to the catalytic domain. One of these, T389 in the shorter form of S6K1, which is located at a hydrophobic motif carboxyterminal to the kinase domain, is directly phosphorylated by mTOR as part of the mTORC1 complex [182, 183]. Phosphorylation here is required for the consequent phosphorylation of S6K1 by PDK1 at a T229 in the activation loop of the catalytic domain. Phosphorylation at T229 allows full activation of S6K1. S6K2 is likely regulated in a similar manner. Both S6K1 and 2 contain a TOS motif within their N-terminus region, which interacts with Raptor, promoting phosphorylation of S6Ks by mTORC1 [184]. The phosphorylation within the C-terminal region seems to open access to the sites T389/T229, phosphorylation of which provides the complete activation. It is not known exactly which kinase is responsible for C-terminal phosphorylation sites. Nevertheless mTOR also indirectly contributes to the phosphorylation of the C-terminal sites. A motif RSPRR exists in this region probably plays a significant role in the inhibitory effect of the C-terminal region. It has been speculated that a negative S6K1 regulator binds S6K via this motif and that mTOR could broke this binding [184]. The C-terminal region of S6K1 also determines whether S6K1 can be phosphorylated by mTORC2. Mutant S6K1 with deletion of this region is a substrate for mTORC2 [185]. Some data indicate that for S6K1 activation, mTOR can directly phosphorylate S371 *in vitro*, and this event modulates T389 phosphorylation by mTOR [186, 187].

In addition to the discussed above mTORC1 targets, S6Ks and 4E-BP1, both of which modulate translation initiation, mTOR signaling also regulates the translation elongation process through the phosphorylation of eukaryotic elongation factor 2 (eEF2). eEF2 is a GTP binding protein that mediates the translocation step of elongation [188]. eEF2 is

phosphorylated at T56 within the GTP-binding domain and this phosphorylation impedes its ability to bind the ribosome, thus inhibiting its function [188, 189]. Insulin and other stimuli induce the dephosphorylation of eEF2, and this effect is blocked by rapamycin, indicating that this effect is also mediated through mTOR [190]. The eEF2 phosphorylation function is attributed to a highly specific enzyme called eEF2 kinase (eEF2K) [190]. Phosphorylation of eEF2 at T56 by eEF2 kinase impedes the eEF2 binding of to the ribosome and the translocation step of the elongation [188]. The calcium/calmodulin-dependent protein kinase eEF2K is an atypical enzyme since the sequence of its catalytic domain differs substantially from that of other protein kinases, and it is not a member, e.g., of the main Ser-Thr-Tyr kinase superfamily [191]. The C-terminal half of the eEF2K polypeptide contains several sites of phosphorylation including the binding site for the substrate eEF2 at the C-terminus [192]. mTOR negatively regulates eEF2 kinase and consequently activates eEF2. mTOR is considered being able to phosphorylate 3 sites, as was determined by their rapamycin- and/or amino-acid starvation sensitivities [188, 193]. S366 in the C terminus of the catalytic domain has been identified as the site being phosphorylated by S6K and by p90RSK [190]. The phosphorylation at S359 has been shown to be also regulated in a rapamycin-sensitive manner in response to insulin-like growth factor 1 (IGF1) and inactivates eEF2K [194], but the kinase responsible for this phosphorylation remains to be determined. Recently, a novel phosphorylation site located immediately adjacent to the CaM-binding site in eEF2K that is regulated markedly in response to insulin in an mTOR dependent manner has been identified. This site (S78) is not known to be phosphorylated by any known protein kinase in the mTOR pathway. Phosphorylation at this third site also causes the inactivation of eEF2 kinase, in this case by inhibiting the binding of CaM, which binds immediately C-terminal to S78 [193]. eEF2K is thought to be a target of signaling from mTOR independently of other known targets of this pathway, which implies the existence of a novel (probably mTORcontrolled) protein kinase that could acts upon S78 in eEF2K. These data provide a molecular mechanism by which mTOR could regulate peptide chain elongation.

Protein Phosphorylation as a Key Mechanism of mTORC1/2 Signaling Pathways 25

**Figure 5. mTORC1 downstream signaling and translation regulation.**

initiation factor 1A and polymerase I respectively.

also stimulates the association between eIF3 and eIF-4G.

**A.** mTOR phosphorylates two major targets: 4E-BP1 and S6Ks. Hypophosphorylated 4E-BP1 binds tightly to eIF-4E, thereby preventing its interaction with eIF-4G and thus inhibiting translation. Phosphorylated 4E-BP1 is released from eIF-4E resulting in the recruitment of eIF-4G to the 5′-cap, and thereby allowing translation initiation to proceed. Phosphorylation of p70S6K stimulates its activity towards several substrates, including 40S ribosomal protein S6, translation initiation factor eIF-4B, elongation factor kinase eEF2K, and PDCD4 protein. Following S6K-mediated phosphorylation, eIF-4B is recruited to the translation pre-initiation complex and enhances the RNA helicase activity of eIF-4A. S6K1dependent phosphorylation of PDCD4 prevents its inhibitory effect towards eIF-4A helicase. mTORC1 also contributes to the translation elongation through the regulation of eEF2. mTOR negatively regulates eEF2 kinase (either directly or via p70S6K activation) and thereby activates eEF2. mTOR signaling also contributes to the regulation of tRNA production, promotion of rRNA synthesis and ribosome biogenesis activating TFIIIC and promoting the association between transcription

**B.** In the absence of extracellular stimuli, S6K1 is associated with eIF3 while 4E-BP1 binding to eIF-4E prevents its interaction with eIF-4G and thus inhibiting translation. In response to extracellular stimuli, such as growth factors or nutrients, the mTOR complex is recruited to eIF3 to phosphorylate S6K1 and 4E-BP1. Phosphorylation and activation of S6K1 leads to its dissociation from eIF3. Activated S6K1 then phosphorylates eIF4B and S6. Phosphorylation of eIF-4B 2 promotes its association with eIF3. mTOR

Since the protein synthesis depends on the amount of ribosomes and transfer RNAs (tRNAs) it is important to know that mTOR signaling also contributes to the regulation of tRNA production, promotion of rRNA synthesis and ribosome biogenesis. Thus mTOR signaling tightly regulates transcription of ribosomal RNAs (rRNAs) and tRNAs by RNA polymerases I and III [195]. mTOR can associate with general transcription factor III C (TFIIIC) and relieve its inhibitor Maf1, leading to increased tRNA production. mTORC1 activity also promotes association between transcription initiation factor 1A (TIF-1A) and polymerase I (PolI), thereby promoting rRNA synthesis [35]. The activity of several other transcription factors, such as signal transducer and activator of transcription-1 and -3 (Stat-1 and Stat-3) is also suggested to be regulated by mTORC1-mediated phosphorylation in a rapamycinsensitive manner [196].

## **4.2. TORC1-mediated repression of autophagy**

Autophagy is a lysosomal-dependent cellular degradation process that generates nutrients and energy to maintain essential cellular activities upon nutrient starvation. A term

Protein Phosphorylation as a Key Mechanism of mTORC1/2 Signaling Pathways 25

#### **Figure 5. mTORC1 downstream signaling and translation regulation.**

24 Protein Phosphorylation in Human Health

sensitive manner [196].

phosphorylated at T56 within the GTP-binding domain and this phosphorylation impedes its ability to bind the ribosome, thus inhibiting its function [188, 189]. Insulin and other stimuli induce the dephosphorylation of eEF2, and this effect is blocked by rapamycin, indicating that this effect is also mediated through mTOR [190]. The eEF2 phosphorylation function is attributed to a highly specific enzyme called eEF2 kinase (eEF2K) [190]. Phosphorylation of eEF2 at T56 by eEF2 kinase impedes the eEF2 binding of to the ribosome and the translocation step of the elongation [188]. The calcium/calmodulin-dependent protein kinase eEF2K is an atypical enzyme since the sequence of its catalytic domain differs substantially from that of other protein kinases, and it is not a member, e.g., of the main Ser-Thr-Tyr kinase superfamily [191]. The C-terminal half of the eEF2K polypeptide contains several sites of phosphorylation including the binding site for the substrate eEF2 at the C-terminus [192]. mTOR negatively regulates eEF2 kinase and consequently activates eEF2. mTOR is considered being able to phosphorylate 3 sites, as was determined by their rapamycin- and/or amino-acid starvation sensitivities [188, 193]. S366 in the C terminus of the catalytic domain has been identified as the site being phosphorylated by S6K and by p90RSK [190]. The phosphorylation at S359 has been shown to be also regulated in a rapamycin-sensitive manner in response to insulin-like growth factor 1 (IGF1) and inactivates eEF2K [194], but the kinase responsible for this phosphorylation remains to be determined. Recently, a novel phosphorylation site located immediately adjacent to the CaM-binding site in eEF2K that is regulated markedly in response to insulin in an mTOR dependent manner has been identified. This site (S78) is not known to be phosphorylated by any known protein kinase in the mTOR pathway. Phosphorylation at this third site also causes the inactivation of eEF2 kinase, in this case by inhibiting the binding of CaM, which binds immediately C-terminal to S78 [193]. eEF2K is thought to be a target of signaling from mTOR independently of other known targets of this pathway, which implies the existence of a novel (probably mTORcontrolled) protein kinase that could acts upon S78 in eEF2K. These data provide a molecular

mechanism by which mTOR could regulate peptide chain elongation.

**4.2. TORC1-mediated repression of autophagy**

Since the protein synthesis depends on the amount of ribosomes and transfer RNAs (tRNAs) it is important to know that mTOR signaling also contributes to the regulation of tRNA production, promotion of rRNA synthesis and ribosome biogenesis. Thus mTOR signaling tightly regulates transcription of ribosomal RNAs (rRNAs) and tRNAs by RNA polymerases I and III [195]. mTOR can associate with general transcription factor III C (TFIIIC) and relieve its inhibitor Maf1, leading to increased tRNA production. mTORC1 activity also promotes association between transcription initiation factor 1A (TIF-1A) and polymerase I (PolI), thereby promoting rRNA synthesis [35]. The activity of several other transcription factors, such as signal transducer and activator of transcription-1 and -3 (Stat-1 and Stat-3) is also suggested to be regulated by mTORC1-mediated phosphorylation in a rapamycin-

Autophagy is a lysosomal-dependent cellular degradation process that generates nutrients and energy to maintain essential cellular activities upon nutrient starvation. A term **A.** mTOR phosphorylates two major targets: 4E-BP1 and S6Ks. Hypophosphorylated 4E-BP1 binds tightly to eIF-4E, thereby preventing its interaction with eIF-4G and thus inhibiting translation. Phosphorylated 4E-BP1 is released from eIF-4E resulting in the recruitment of eIF-4G to the 5′-cap, and thereby allowing translation initiation to proceed. Phosphorylation of p70S6K stimulates its activity towards several substrates, including 40S ribosomal protein S6, translation initiation factor eIF-4B, elongation factor kinase eEF2K, and PDCD4 protein. Following S6K-mediated phosphorylation, eIF-4B is recruited to the translation pre-initiation complex and enhances the RNA helicase activity of eIF-4A. S6K1dependent phosphorylation of PDCD4 prevents its inhibitory effect towards eIF-4A helicase. mTORC1 also contributes to the translation elongation through the regulation of eEF2. mTOR negatively regulates eEF2 kinase (either directly or via p70S6K activation) and thereby activates eEF2. mTOR signaling also contributes to the regulation of tRNA production, promotion of rRNA synthesis and ribosome biogenesis activating TFIIIC and promoting the association between transcription initiation factor 1A and polymerase I respectively.

**B.** In the absence of extracellular stimuli, S6K1 is associated with eIF3 while 4E-BP1 binding to eIF-4E prevents its interaction with eIF-4G and thus inhibiting translation. In response to extracellular stimuli, such as growth factors or nutrients, the mTOR complex is recruited to eIF3 to phosphorylate S6K1 and 4E-BP1. Phosphorylation and activation of S6K1 leads to its dissociation from eIF3. Activated S6K1 then phosphorylates eIF4B and S6. Phosphorylation of eIF-4B 2 promotes its association with eIF3. mTOR also stimulates the association between eIF3 and eIF-4G.

autophagy appeared from Greek "auto" (self) and "phagy" (to eat), refers to an evolutionarily conserved, multi-step lysosomal degradation process in which a cell degrades long-lived proteins and damaged organelles. Three forms of autophagy have been identified, namely macroautophagy, microautophagy and chaperone-mediated autophagy that differ with respect to their modes of delivery to lysosome and physiological functions [197]. Macroautophagy (hereafter autophagy) is the major regulated catabolic mechanism that involves the delivery of cytosolic cargo sequestered inside specific intracellular doublemembrane vesicles, called autophagosomes to the lysosomal compartment and subsequent fusion with lysosomes to form single-membrane-bound autophagolysosomes, in which the sequestered material is degraded by acidic lysosomal hydrolases. On one hand, autophagy is crucial for cell survival under extreme conditions through degradation of intracellular macromolecules, which provides the energy required for minimal cell functioning when nutrients are deprived or scarce. Also, autophagy-mediated elimination of altered cytosolic constituents, such as aggregated proteins or damaged organelles, preserves cells from further damages, indicating that autophagy plays a protective role in early stages of cancer [198]. On the other hand, autophagy plays a death-promoting role as type II programmed cell death (type II PCD), compared to apoptosis (type I PCD), as a bona fide tumor suppressor mechanism in cancer [199].

Protein Phosphorylation as a Key Mechanism of mTORC1/2 Signaling Pathways 27

Atg13 and FIP200. Several serine and threonine residues in human Ulk1 whose phosphorylation was decreased after starvation have been recently identified from which S638 and S758 have been proposed to be most probable mTORC1 negative phosphorylation sites [203, 206]. Ulk1/2 autophosphorylation and following FIP200 and Atg13 phosphorylation in turn leads to translocation of the entire complex to the preautophagosomal membrane and to autophagy induction [200, 201, 205]. However, the functional relevance of Ulk1/2-mediated phosphorylation of Atg13 and FIP200 for this recruitment and the relevant phosphorylation sites has not been verified yet. Interestingly, another Ulk1-dependent phosphorylation site in human Atg13 (S318) has been identified recently [207]. The authors of that study could show that the Hsp90-Cdc37 chaperone

**Figure 6. mTORC1 downstream signaling and autophagy regulation.** Ulk1 and Ulk2 form a stable complex with Atg13, FIP200 and Atg101. Under fed conditions mTORC1 phosphorylates Ulk1/2 and Atg13, thereby inhibiting the Ulk1/2 kinase activity and complex stability. In response to starvation, the mTORC1-dependent phosphorylation sites in Ulk1/2 are rapidly dephosphorylated, and Ulk1/2 autophosphorylates and phosphorylates Atg13 and FIP200 resulting in translocation of the entire complex to the pre-autophagosomal membrane and autophagy induction. Alternatively, Ulk1/2 is phosphorylated by AMPK and thereby activated. In addition, AMPK indirectly leads to the induction of

In yeast, autophagosomes originate from a single preautophagosomal structure. Although an equivalent structure seems to be absent from mammalian cells, a special subdomain in the endoplasmic reticulum (ER) termed the "omegasome" has been suggested as a putative origin of autophagosomes. This structure is enriched in PI(3)P, a product of the phosphatidylinositol 3-kinase (PI3K). A hierarchical analysis of the mammalian Atg proteins could recently confirm the recruitment of Ulk1 proximal to these omegasomes [208]. The translocation of Ulk1, presumably in a complex with Atg13 and FIP200, is the initial step of autophagosome biogenesis and is completely abrogated in *FIP200–*/– cells [208]. The subsequent recruitment of the PI3K depends on Ulk1 and its kinase activity [208]. Recently, two groups found evidence for the mechanism by which Ulk1 and Ulk2 in turn negatively regulate mTORC1 signaling. Particularly, the phosphorylation of Raptor at numerous sites

autophagy by inhibiting mTORC1 through phosphorylation of Raptor or TSC2.

complex selectively stabilizes and activates Ulk1.

The ability of mTORC1 to regulate autophagy is as highly conserved as well as the process of autophagy itself. AMPK has been indicated as a main upstream regulator of mTORC1 mediated autophagy inhibition.

The mechanism by which TORC1 negatively regulates the autophagic machinery has first been described in yeast. Genetic screenings for autophagy defective mutants led to the identification of more than 30 essential autophagy-related genes (Atg).These proteins can be classified into several groups depending on their function and interdependency. Most upstream is a protein complex that comprises the serine/threonine kinase Atg1, as well as two accessory proteins Atg13 and Atg17. In mammals, two homologs of Atg1, uncoordinated 51-like kinase 1 (ULK1) and ULK2 have been identified. Accumulating evidence suggests that ULK1 is a key regulator of autophagy initiation. ULK1 is directly phosphorylated by TORC1 [200-202]. Recently, it has also been shown that mTORC1 mediated phosphorylation of ULK1 impairs its activation by AMPK and results in an overall decrease in autophagy [203]. ULK1 and ULK2 are found in a stable complex with mammalian autophagy-related protein 13 (mAtg13), the scaffold protein FAK-family interacting protein of 200 kDa (FIP200) [204] [201] and Atg101, an additional binding partner of Atg13 that has no ortholog in yeast [205]. In contrast to yeast, the composition of this complex does not change with the nutrient status. Several data evidence that the phosphorylation status within the Ulk1/2-Atg13-FIP200 complex dramatically changes with the nutrient availability. Under rich growth conditions, mTORC1 associates with the Ulk1/2- Atg13-FIP200 complex, via direct interaction between Raptor and Ulk1/2 (37). The active mTOR phosphorylates Atg13 and Ulk1/2 [201], thereby downregulating Ulk1/2 kinase activity and suppressing autophagy **(Figure 6)**. In response to starvation, the mTORC1 dependent phosphorylation sites in Ulk1/2 are rapidly dephosphorylated by yet unknown phosphatases, what stimulates Ulk1/2 autophosphorylation and phosphorylation of both Atg13 and FIP200. Several serine and threonine residues in human Ulk1 whose phosphorylation was decreased after starvation have been recently identified from which S638 and S758 have been proposed to be most probable mTORC1 negative phosphorylation sites [203, 206]. Ulk1/2 autophosphorylation and following FIP200 and Atg13 phosphorylation in turn leads to translocation of the entire complex to the preautophagosomal membrane and to autophagy induction [200, 201, 205]. However, the functional relevance of Ulk1/2-mediated phosphorylation of Atg13 and FIP200 for this recruitment and the relevant phosphorylation sites has not been verified yet. Interestingly, another Ulk1-dependent phosphorylation site in human Atg13 (S318) has been identified recently [207]. The authors of that study could show that the Hsp90-Cdc37 chaperone complex selectively stabilizes and activates Ulk1.

26 Protein Phosphorylation in Human Health

suppressor mechanism in cancer [199].

mediated autophagy inhibition.

autophagy appeared from Greek "auto" (self) and "phagy" (to eat), refers to an evolutionarily conserved, multi-step lysosomal degradation process in which a cell degrades long-lived proteins and damaged organelles. Three forms of autophagy have been identified, namely macroautophagy, microautophagy and chaperone-mediated autophagy that differ with respect to their modes of delivery to lysosome and physiological functions [197]. Macroautophagy (hereafter autophagy) is the major regulated catabolic mechanism that involves the delivery of cytosolic cargo sequestered inside specific intracellular doublemembrane vesicles, called autophagosomes to the lysosomal compartment and subsequent fusion with lysosomes to form single-membrane-bound autophagolysosomes, in which the sequestered material is degraded by acidic lysosomal hydrolases. On one hand, autophagy is crucial for cell survival under extreme conditions through degradation of intracellular macromolecules, which provides the energy required for minimal cell functioning when nutrients are deprived or scarce. Also, autophagy-mediated elimination of altered cytosolic constituents, such as aggregated proteins or damaged organelles, preserves cells from further damages, indicating that autophagy plays a protective role in early stages of cancer [198]. On the other hand, autophagy plays a death-promoting role as type II programmed cell death (type II PCD), compared to apoptosis (type I PCD), as a bona fide tumor

The ability of mTORC1 to regulate autophagy is as highly conserved as well as the process of autophagy itself. AMPK has been indicated as a main upstream regulator of mTORC1

The mechanism by which TORC1 negatively regulates the autophagic machinery has first been described in yeast. Genetic screenings for autophagy defective mutants led to the identification of more than 30 essential autophagy-related genes (Atg).These proteins can be classified into several groups depending on their function and interdependency. Most upstream is a protein complex that comprises the serine/threonine kinase Atg1, as well as two accessory proteins Atg13 and Atg17. In mammals, two homologs of Atg1, uncoordinated 51-like kinase 1 (ULK1) and ULK2 have been identified. Accumulating evidence suggests that ULK1 is a key regulator of autophagy initiation. ULK1 is directly phosphorylated by TORC1 [200-202]. Recently, it has also been shown that mTORC1 mediated phosphorylation of ULK1 impairs its activation by AMPK and results in an overall decrease in autophagy [203]. ULK1 and ULK2 are found in a stable complex with mammalian autophagy-related protein 13 (mAtg13), the scaffold protein FAK-family interacting protein of 200 kDa (FIP200) [204] [201] and Atg101, an additional binding partner of Atg13 that has no ortholog in yeast [205]. In contrast to yeast, the composition of this complex does not change with the nutrient status. Several data evidence that the phosphorylation status within the Ulk1/2-Atg13-FIP200 complex dramatically changes with the nutrient availability. Under rich growth conditions, mTORC1 associates with the Ulk1/2- Atg13-FIP200 complex, via direct interaction between Raptor and Ulk1/2 (37). The active mTOR phosphorylates Atg13 and Ulk1/2 [201], thereby downregulating Ulk1/2 kinase activity and suppressing autophagy **(Figure 6)**. In response to starvation, the mTORC1 dependent phosphorylation sites in Ulk1/2 are rapidly dephosphorylated by yet unknown phosphatases, what stimulates Ulk1/2 autophosphorylation and phosphorylation of both

**Figure 6. mTORC1 downstream signaling and autophagy regulation.** Ulk1 and Ulk2 form a stable complex with Atg13, FIP200 and Atg101. Under fed conditions mTORC1 phosphorylates Ulk1/2 and Atg13, thereby inhibiting the Ulk1/2 kinase activity and complex stability. In response to starvation, the mTORC1-dependent phosphorylation sites in Ulk1/2 are rapidly dephosphorylated, and Ulk1/2 autophosphorylates and phosphorylates Atg13 and FIP200 resulting in translocation of the entire complex to the pre-autophagosomal membrane and autophagy induction. Alternatively, Ulk1/2 is phosphorylated by AMPK and thereby activated. In addition, AMPK indirectly leads to the induction of autophagy by inhibiting mTORC1 through phosphorylation of Raptor or TSC2.

In yeast, autophagosomes originate from a single preautophagosomal structure. Although an equivalent structure seems to be absent from mammalian cells, a special subdomain in the endoplasmic reticulum (ER) termed the "omegasome" has been suggested as a putative origin of autophagosomes. This structure is enriched in PI(3)P, a product of the phosphatidylinositol 3-kinase (PI3K). A hierarchical analysis of the mammalian Atg proteins could recently confirm the recruitment of Ulk1 proximal to these omegasomes [208]. The translocation of Ulk1, presumably in a complex with Atg13 and FIP200, is the initial step of autophagosome biogenesis and is completely abrogated in *FIP200–*/– cells [208]. The subsequent recruitment of the PI3K depends on Ulk1 and its kinase activity [208]. Recently, two groups found evidence for the mechanism by which Ulk1 and Ulk2 in turn negatively regulate mTORC1 signaling. Particularly, the phosphorylation of Raptor at numerous sites

was strongly enhanced after overexpression of Ulk1. Interestingly, one of these residues (T792) is the abovementioned effector site through which AMPK negatively regulates mTORC1 activity [45]. The multiple Ulk1-dependent phosphorylation of Raptor either results in direct inhibition of mTORC1 kinase activity [209], or interferes with Raptorsubstrate interaction [210], thus finally leads to reduced phosphorylation of mTORC1 downstream targets.

Protein Phosphorylation as a Key Mechanism of mTORC1/2 Signaling Pathways 29

The best-characterized target of mTORC2 is AKT, which is phosphorylated at S473 upon TORC2 activation [53, 58, 85]. Numerous studies attempted to identify the crucial kinase(s) (often referred to as PDK2) responsible for the phosphorylation of S473 in Akt. Several enzymes are in the candidate list, including PDK1, integrin-linked kinase (ILK), Akt itself, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and mTORC2 [215]. Since mTORC2 complex fulfills the role of the Akt S473 kinase, mTORC2 has been identified as the PDK2 [85]. Akt is a member of the AGC kinase family (see above), which also includes S6Ks, serum glucocorticoid-induced protein kinase (SGKs), RSKs, and PKCs [62, 216]. mTORC2 has been shown to phosphorylate AKT, SGK and PKC [85]. mTORC2 seems to regulate Akt by phosphorylation of its two different sites. The mTORC2-mediated Akt hydrophobic motif phosphorylation on the regulatory S473 site is dependent on growth factor signaling, whereas a basal activity of mTORC2 maintains the constitutive phosphorylation of Akt on T450 site in its turn motif [217]. This difference indicates that phosphorylation of the T450 and S473 sites on Akt by mTORC2 are separate events and might take place at different locations. It has been proposed that translocation of Akt to the plasma membrane coupled with its phosphorylation on T308 and S473 is a critical step in activation of Akt by growth factor signaling [217]. Phosphorylation of AKT on S473 enhances the activation phosphorylation motif at T308, which is absolutely required for

The major functions of mTORC2 include the regulation of cytoskeletal organization and the promotion of cell survival. If the last one is mediated apparently through AKT activation, the mechanisms, which realize mTORC2 function in cytoskeletal reorganization, are not obvious. Paxillin, which functions as a docking protein, localizing to the focal adhesions of adherent cells [218] has been shown to be highly phosphorylated at Tyr118. Knockdown of mTORC2 inhibited the phosphorylation of paxillin [3]. Rho, Rac and Cdc42, three bestcharacterized members of the Rho family of small GTPases, were demonstrated to be involved in actin cytoskeleton assembly and disassembly [219]. It was reported that mTORC2 may function as upstream regulator of Rho GTPases to regulate the actin

Interestingly, the TORC2-mediated activation of AKT places TORC2 upstream of TORC1 in the TOR signaling cascade. A most recent publication has highlighted a role for ribosomes in the activation of TORC2 [38]. The authors have found that active mTORC2 was physically associated with the ribosome, and insulin-stimulated PI3K signaling promoted mTORC2 ribosome binding. Interaction of mTORC2 with NIP7 (nuclear import 7, a protein responsible for ribosome biogenesis and rRNA maturation) was shown to be required for full activation of mTORC2 by insulin. Noteworthy, inhibition of protein translation had no effect on mTORC2 activation, supporting the notion that mTORC2 is activated by the ribosome, but not translation. Ribosome associated mTORC2 displays kinase activity toward AKT *in vitro*. Inhibition of PI3K activity blocks the interaction between the ribosome and mTORC2, as well as inhibits mTORC2 activation in response to insulin, confirming that NIP7-ribosome assembly activates mTORC2 downstream from PI3K. It appears that the mTORC2 components, Rictor and/or Sin1, which are not found in mTORC1, interact with

AKT activity.

cytoskeleton [3].

Apart from mTOR, Ulk1/2 is phosphorylated (probably on S317 and S777 or S555 according to different studies) by AMPK under glucose starvation and thereby activated [112, 203, 211, 212]. Under nutrient sufficiency phosphorylation of ULK1 S758 by active mTORC1 disrupts ULK1 interaction with, and hence activation by, AMPK [203]. Although the data concerning the role of ULK1/2 certain sites phosphorylation is rather discrepant it is clear that in mammals, phosphorylation of ULK1 by AMPK is strongly required for ULK function in the response to nutrient deprivation. Therefore, AMPK could control ULK1 via a two-pronged mechanism, ensuring activation only under the appropriate cellular conditions – firstly, by direct phosphorylation and secondly, by suppression of mTORC1-mediated ULK1 inhibition [212]. Several studies demonstrated that Ulk1 in addition directly interferes with mTORC1 downstream signaling and negatively regulates S6K1 activity, both in *Drosophila*  and mammalian cells [213]. Taken together these data evidence that mTOR subnetwork occupy the key position in autophagic pathways.

## **5. Signaling up and downstream of mTORC2**

In contrast to mTORC1, very little is known about the upstream regulation of TORC2. Rapamycin–FKBP12 complex does not bind directly to mTORC2, but long-term rapamycin treatment disrupts mTORC2 assembly in ~20% of cancer cell lines through an unknown mechanism [56]. It remains to be determined why rapamycin-mediated inhibition of mTORC2 assembly only occurs in certain cell types. One hypothesis suggests that some mTORC2 subunits could prevent the binding of rapamycin/FKBP12 complex to the mTOR FRB domain by the competing mechanism (reviewed in [62]). However, there are no enough data to support this model.

It seems that mTORC2 is activated in response to growth factors but is insensitive to nutrients and energetic stress, [214]. Thus, like TORC1, TORC2 can be stimulated by growth factors through PI3K [3]. Consequently, treatment with PI3K inhibitors can inhibit TORC2 mediated target phosphorylation [85]. Thus it was suggested that mTORC2 lies downstream of PI3K signaling [85]. Rheb which is known as a key upstream activator of mTORC1 showed negative and indirect effect on the regulation of mTORC2 both in *Drosophila* and mammalian cells [59]. Some data pointed on TSC1-TSC2 function in mTORC2 regulation [92]. Moreover, the TSC1/TSC2 complex was found to physically associate with mTORC2, but not with mTORC1. The molecular mechanism through which the TSC1/TSC2 complex promotes mTORC2 activation is unclear. It is also currently unknown whether some pathways that regulate TSC1/TSC2 ability to inhibit mTORC1, also influence on mTORC2 activation.

The best-characterized target of mTORC2 is AKT, which is phosphorylated at S473 upon TORC2 activation [53, 58, 85]. Numerous studies attempted to identify the crucial kinase(s) (often referred to as PDK2) responsible for the phosphorylation of S473 in Akt. Several enzymes are in the candidate list, including PDK1, integrin-linked kinase (ILK), Akt itself, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and mTORC2 [215]. Since mTORC2 complex fulfills the role of the Akt S473 kinase, mTORC2 has been identified as the PDK2 [85]. Akt is a member of the AGC kinase family (see above), which also includes S6Ks, serum glucocorticoid-induced protein kinase (SGKs), RSKs, and PKCs [62, 216]. mTORC2 has been shown to phosphorylate AKT, SGK and PKC [85]. mTORC2 seems to regulate Akt by phosphorylation of its two different sites. The mTORC2-mediated Akt hydrophobic motif phosphorylation on the regulatory S473 site is dependent on growth factor signaling, whereas a basal activity of mTORC2 maintains the constitutive phosphorylation of Akt on T450 site in its turn motif [217]. This difference indicates that phosphorylation of the T450 and S473 sites on Akt by mTORC2 are separate events and might take place at different locations. It has been proposed that translocation of Akt to the plasma membrane coupled with its phosphorylation on T308 and S473 is a critical step in activation of Akt by growth factor signaling [217]. Phosphorylation of AKT on S473 enhances the activation phosphorylation motif at T308, which is absolutely required for AKT activity.

28 Protein Phosphorylation in Human Health

occupy the key position in autophagic pathways.

data to support this model.

activation.

**5. Signaling up and downstream of mTORC2**

downstream targets.

was strongly enhanced after overexpression of Ulk1. Interestingly, one of these residues (T792) is the abovementioned effector site through which AMPK negatively regulates mTORC1 activity [45]. The multiple Ulk1-dependent phosphorylation of Raptor either results in direct inhibition of mTORC1 kinase activity [209], or interferes with Raptorsubstrate interaction [210], thus finally leads to reduced phosphorylation of mTORC1

Apart from mTOR, Ulk1/2 is phosphorylated (probably on S317 and S777 or S555 according to different studies) by AMPK under glucose starvation and thereby activated [112, 203, 211, 212]. Under nutrient sufficiency phosphorylation of ULK1 S758 by active mTORC1 disrupts ULK1 interaction with, and hence activation by, AMPK [203]. Although the data concerning the role of ULK1/2 certain sites phosphorylation is rather discrepant it is clear that in mammals, phosphorylation of ULK1 by AMPK is strongly required for ULK function in the response to nutrient deprivation. Therefore, AMPK could control ULK1 via a two-pronged mechanism, ensuring activation only under the appropriate cellular conditions – firstly, by direct phosphorylation and secondly, by suppression of mTORC1-mediated ULK1 inhibition [212]. Several studies demonstrated that Ulk1 in addition directly interferes with mTORC1 downstream signaling and negatively regulates S6K1 activity, both in *Drosophila*  and mammalian cells [213]. Taken together these data evidence that mTOR subnetwork

In contrast to mTORC1, very little is known about the upstream regulation of TORC2. Rapamycin–FKBP12 complex does not bind directly to mTORC2, but long-term rapamycin treatment disrupts mTORC2 assembly in ~20% of cancer cell lines through an unknown mechanism [56]. It remains to be determined why rapamycin-mediated inhibition of mTORC2 assembly only occurs in certain cell types. One hypothesis suggests that some mTORC2 subunits could prevent the binding of rapamycin/FKBP12 complex to the mTOR FRB domain by the competing mechanism (reviewed in [62]). However, there are no enough

It seems that mTORC2 is activated in response to growth factors but is insensitive to nutrients and energetic stress, [214]. Thus, like TORC1, TORC2 can be stimulated by growth factors through PI3K [3]. Consequently, treatment with PI3K inhibitors can inhibit TORC2 mediated target phosphorylation [85]. Thus it was suggested that mTORC2 lies downstream of PI3K signaling [85]. Rheb which is known as a key upstream activator of mTORC1 showed negative and indirect effect on the regulation of mTORC2 both in *Drosophila* and mammalian cells [59]. Some data pointed on TSC1-TSC2 function in mTORC2 regulation [92]. Moreover, the TSC1/TSC2 complex was found to physically associate with mTORC2, but not with mTORC1. The molecular mechanism through which the TSC1/TSC2 complex promotes mTORC2 activation is unclear. It is also currently unknown whether some pathways that regulate TSC1/TSC2 ability to inhibit mTORC1, also influence on mTORC2 The major functions of mTORC2 include the regulation of cytoskeletal organization and the promotion of cell survival. If the last one is mediated apparently through AKT activation, the mechanisms, which realize mTORC2 function in cytoskeletal reorganization, are not obvious. Paxillin, which functions as a docking protein, localizing to the focal adhesions of adherent cells [218] has been shown to be highly phosphorylated at Tyr118. Knockdown of mTORC2 inhibited the phosphorylation of paxillin [3]. Rho, Rac and Cdc42, three bestcharacterized members of the Rho family of small GTPases, were demonstrated to be involved in actin cytoskeleton assembly and disassembly [219]. It was reported that mTORC2 may function as upstream regulator of Rho GTPases to regulate the actin cytoskeleton [3].

Interestingly, the TORC2-mediated activation of AKT places TORC2 upstream of TORC1 in the TOR signaling cascade. A most recent publication has highlighted a role for ribosomes in the activation of TORC2 [38]. The authors have found that active mTORC2 was physically associated with the ribosome, and insulin-stimulated PI3K signaling promoted mTORC2 ribosome binding. Interaction of mTORC2 with NIP7 (nuclear import 7, a protein responsible for ribosome biogenesis and rRNA maturation) was shown to be required for full activation of mTORC2 by insulin. Noteworthy, inhibition of protein translation had no effect on mTORC2 activation, supporting the notion that mTORC2 is activated by the ribosome, but not translation. Ribosome associated mTORC2 displays kinase activity toward AKT *in vitro*. Inhibition of PI3K activity blocks the interaction between the ribosome and mTORC2, as well as inhibits mTORC2 activation in response to insulin, confirming that NIP7-ribosome assembly activates mTORC2 downstream from PI3K. It appears that the mTORC2 components, Rictor and/or Sin1, which are not found in mTORC1, interact with

the 60S subunit of ribosome. Interestingly, another study [220] has also recently reported the association of mTORC2 with the ribosome and proposed that the ribosomal association is important for the cotranslational phosphorylation of the AKT turn motif. These findings are coherent with very recent data that point on endoplasmic reticulum (ER), the cellular organelle highly enriched with ribosomes, as a major compartment of mTORC2 localization. Moreover, the signaling from growth factor does not change the ER localization of mTORC2 as well as its translocation to the plasma membrane. Besides it was suggested that the mTORC2-dependent phosphorylation of Akt on S473 occurs on the surface of ER [37]. These observations raise many interesting questions regarding the regulation of TORC2 and its ribosomal interactions, but it also indicates that additional levels of interplay between TORC2 and TORC1 may exist, as both complexes are linked to the process of ribosome biogenesis.

Protein Phosphorylation as a Key Mechanism of mTORC1/2 Signaling Pathways 31

(S221, S363, S380 and T573 in human RSK1). Following mitogen stimulation, ERK1/2 phosphorylates T573 at the C-terminal domain (CTKD) activation loop of RSK, resulting in CTKD activation. ERK1/2 might also phosphorylate S363 (the turn motif) and T359 (unknown function) in the RSK linker region. The activated CTKD of RSK then autophosphorylates S380 in the hydrophobic motif, creating a docking site for PDK1. After binding, PDK1 phosphorylates the NTKD activation loop S221, leading to the complete activation of the protein and following phosphorylation of the substrates by the NTKD. The NTKD also phosphorylates S749 in the CTKD domain of RSK, differentially modulating the interaction of RSK isoforms with ERK1/2 and thereby completing a sequence of coordinated phosphorylation events and protein–protein interactions that culminate in RSK activation and downstream signaling throughout the cell [224]. Other factors that have been shown to be involved in the activation of RSK include the p38 MAPK, the ERK5 MAPK and fibroblast growth factor receptor-3 (FGFR3). RSK was found to phosphorylate TSC2 at the C-terminus S1798 [225] and, to a lesser extent, the two conserved Akt sites (S939 and T1462) and inactivates its suppressor function, thereby promoting mTOR signaling and translation (**Figure 7)**. RSK mediated phosphorylation of TSC2 is additive to AKT mediated inhibitory modifications of TSC2, but how these phosphorylation events lead to TSC2

Erk1/2 kinase itself also impacts the mTORC1 regulation. Thus, a number of additional sites on TSC2 were found to be weakly induced by PMA [89], including an ERK consensus site S664. This site, and a second site on TSC2, S540, was independently found to be directly phosphorylated by ERK and to contribute to ERK-mediated activation of mTORC1 signaling [226]. Strikingly, phosphorylation of S540 and/or S664 by ERK was found to disrupt the association between TSC1 and TSC2. This effect was also detected following phosphorylation of the TSC1/TSC2 complex *in vitro,* suggesting that it is direct and does not

In addition, it was recently shown that RSK also directly impacts the mTORC1 complex phosphorylating Raptor, and thereby promotes mTORC1 kinase activity [46]. RSK phosphorylates at least two evolutionarily conserved Raptor serine residues that lie within a region with no homology to known functional domains. Whereas S721 lies within a classical RSK consensus sequence (RXRXXpS/T), S719 is located within a minimal phosphoacceptor sequence (RXXpS) that was found to be sufficient in other RSK substrates, such as DAPK, c-Fos, and CREB. Although the underlying molecular mechanism of this was not fully defined, this study provided new insights into Ras–ERK signals to mTORC1. As tumor promoting phorbol esters and some growth factors activate mTORC1 signaling independently of AKT, phosphorylation of Raptor by RSK might provide a mechanism to overcome the inhibitory effects of PRAS40 inhibitory phosphorylation of TSC2 at S664 and S1798, respectively [89, 226, 227]. Collectively, these data suggest that ERK signaling activates mTORC1 through multisite phosphorylation events by both ERK and its

Erk1/2-RSK pathway also contributes to the mTORC1 downstream signaling, this includes RSK dependent *in vivo* and *in vitro* phosphorylation of eukaryotic translation initiation

inhibition remains unclear.

require other proteins.

downstream target RSK.

## **6. Crosstalk of mTORC1/2 and major cytokine signaling pathways**

## **6.1. mTORC1/2 and Ras-MAP kinases pathways**

#### *Ras-Erk-RSKs*

In addition to the PI3K–Akt pathway, activation of Ras-MAPK signaling can also stimulate mTORC1 activity. The Ras–mitogen-activated protein kinase (MAPK) pathway is a key signaling pathway that is involved in the regulation of normal cell proliferation, survival, growth and differentiation. This pathway includes the whole number of kinases, being regulated through phosphorylation in consecutive order. The Ras–MAPK signaling network has been the subject of intense research because mutations in (or overexpression of) many of the signaling components from this pathway are a hallmark of several human cancers and other human diseases [221]. The Ras–ERK (extracellular signal-regulated kinase-1 and -2) pathway has an established role in regulating transcription [222], but a connection between this pathway and translational regulation is less clear. Over the past few years, mitogen activated Ras–ERK pathway has been shown to trigger the activation of mTORC1 signaling. This is mediated by ERK and RSK dependent phosphorylations of TORC1 pathway components.

p90RSKs (also known as MAPKAP kinase 1 (mitogen-activated protein kinase-activated protein) kinase-1) are a family of Ser/Thr kinases that lies downstream of the Ras–MAPK cascade and has overlapping substrate specificity with Akt. The RSK isoforms are directly activated by ERK1/2 in response to growth factors, many polypeptide hormones, neurotransmitters, chemokines and other stimuli. RSKs phosphorylate several cytosolic and nuclear targets and they are involved in the regulation of different cellular processes, including cell survival, cell proliferation, cell growth and motility. Following the stimulation of cells with growth factors, RSKs are phosphorylated at multiple Ser and Thr residues by several kinases; these phosphorylation events are directly or indirectly initiated by the activation of the ERK/MAPK cascade [223]. Six different phosphorylation sites have been mapped in RSK1/2 (and are conserved in RSK3/4), of which four have been shown to be important for their activity (S221, S363, S380 and T573 in human RSK1). Following mitogen stimulation, ERK1/2 phosphorylates T573 at the C-terminal domain (CTKD) activation loop of RSK, resulting in CTKD activation. ERK1/2 might also phosphorylate S363 (the turn motif) and T359 (unknown function) in the RSK linker region. The activated CTKD of RSK then autophosphorylates S380 in the hydrophobic motif, creating a docking site for PDK1. After binding, PDK1 phosphorylates the NTKD activation loop S221, leading to the complete activation of the protein and following phosphorylation of the substrates by the NTKD. The NTKD also phosphorylates S749 in the CTKD domain of RSK, differentially modulating the interaction of RSK isoforms with ERK1/2 and thereby completing a sequence of coordinated phosphorylation events and protein–protein interactions that culminate in RSK activation and downstream signaling throughout the cell [224]. Other factors that have been shown to be involved in the activation of RSK include the p38 MAPK, the ERK5 MAPK and fibroblast growth factor receptor-3 (FGFR3). RSK was found to phosphorylate TSC2 at the C-terminus S1798 [225] and, to a lesser extent, the two conserved Akt sites (S939 and T1462) and inactivates its suppressor function, thereby promoting mTOR signaling and translation (**Figure 7)**. RSK mediated phosphorylation of TSC2 is additive to AKT mediated inhibitory modifications of TSC2, but how these phosphorylation events lead to TSC2 inhibition remains unclear.

30 Protein Phosphorylation in Human Health

biogenesis.

*Ras-Erk-RSKs*

components.

the 60S subunit of ribosome. Interestingly, another study [220] has also recently reported the association of mTORC2 with the ribosome and proposed that the ribosomal association is important for the cotranslational phosphorylation of the AKT turn motif. These findings are coherent with very recent data that point on endoplasmic reticulum (ER), the cellular organelle highly enriched with ribosomes, as a major compartment of mTORC2 localization. Moreover, the signaling from growth factor does not change the ER localization of mTORC2 as well as its translocation to the plasma membrane. Besides it was suggested that the mTORC2-dependent phosphorylation of Akt on S473 occurs on the surface of ER [37]. These observations raise many interesting questions regarding the regulation of TORC2 and its ribosomal interactions, but it also indicates that additional levels of interplay between TORC2 and TORC1 may exist, as both complexes are linked to the process of ribosome

**6. Crosstalk of mTORC1/2 and major cytokine signaling pathways**

In addition to the PI3K–Akt pathway, activation of Ras-MAPK signaling can also stimulate mTORC1 activity. The Ras–mitogen-activated protein kinase (MAPK) pathway is a key signaling pathway that is involved in the regulation of normal cell proliferation, survival, growth and differentiation. This pathway includes the whole number of kinases, being regulated through phosphorylation in consecutive order. The Ras–MAPK signaling network has been the subject of intense research because mutations in (or overexpression of) many of the signaling components from this pathway are a hallmark of several human cancers and other human diseases [221]. The Ras–ERK (extracellular signal-regulated kinase-1 and -2) pathway has an established role in regulating transcription [222], but a connection between this pathway and translational regulation is less clear. Over the past few years, mitogen activated Ras–ERK pathway has been shown to trigger the activation of mTORC1 signaling. This is mediated by ERK and RSK dependent phosphorylations of TORC1 pathway

p90RSKs (also known as MAPKAP kinase 1 (mitogen-activated protein kinase-activated protein) kinase-1) are a family of Ser/Thr kinases that lies downstream of the Ras–MAPK cascade and has overlapping substrate specificity with Akt. The RSK isoforms are directly activated by ERK1/2 in response to growth factors, many polypeptide hormones, neurotransmitters, chemokines and other stimuli. RSKs phosphorylate several cytosolic and nuclear targets and they are involved in the regulation of different cellular processes, including cell survival, cell proliferation, cell growth and motility. Following the stimulation of cells with growth factors, RSKs are phosphorylated at multiple Ser and Thr residues by several kinases; these phosphorylation events are directly or indirectly initiated by the activation of the ERK/MAPK cascade [223]. Six different phosphorylation sites have been mapped in RSK1/2 (and are conserved in RSK3/4), of which four have been shown to be important for their activity

**6.1. mTORC1/2 and Ras-MAP kinases pathways**

Erk1/2 kinase itself also impacts the mTORC1 regulation. Thus, a number of additional sites on TSC2 were found to be weakly induced by PMA [89], including an ERK consensus site S664. This site, and a second site on TSC2, S540, was independently found to be directly phosphorylated by ERK and to contribute to ERK-mediated activation of mTORC1 signaling [226]. Strikingly, phosphorylation of S540 and/or S664 by ERK was found to disrupt the association between TSC1 and TSC2. This effect was also detected following phosphorylation of the TSC1/TSC2 complex *in vitro,* suggesting that it is direct and does not require other proteins.

In addition, it was recently shown that RSK also directly impacts the mTORC1 complex phosphorylating Raptor, and thereby promotes mTORC1 kinase activity [46]. RSK phosphorylates at least two evolutionarily conserved Raptor serine residues that lie within a region with no homology to known functional domains. Whereas S721 lies within a classical RSK consensus sequence (RXRXXpS/T), S719 is located within a minimal phosphoacceptor sequence (RXXpS) that was found to be sufficient in other RSK substrates, such as DAPK, c-Fos, and CREB. Although the underlying molecular mechanism of this was not fully defined, this study provided new insights into Ras–ERK signals to mTORC1. As tumor promoting phorbol esters and some growth factors activate mTORC1 signaling independently of AKT, phosphorylation of Raptor by RSK might provide a mechanism to overcome the inhibitory effects of PRAS40 inhibitory phosphorylation of TSC2 at S664 and S1798, respectively [89, 226, 227]. Collectively, these data suggest that ERK signaling activates mTORC1 through multisite phosphorylation events by both ERK and its downstream target RSK.

Erk1/2-RSK pathway also contributes to the mTORC1 downstream signaling, this includes RSK dependent *in vivo* and *in vitro* phosphorylation of eukaryotic translation initiation

factor-4B (eIF4B) and rpS6 [180]. Although early studies indicated S6K1 and S6K2 as the major rpS6 kinases in somatic cells [228, 229], the role of RSK in regulating site-specific rpS6 phosphorylation and translation in somatic cells has been recently readdressed [230]. Particularly, *in vitro* and *in vivo* evidence suggests that S6Ks phosphorylate every site in rpS6, while RSK predominantly phosphorylates S235 and S236 [230]. Studies from *S6k1*/*S6k2*-knockout mice showed that there was minimal phosphorylation of rpS6 at S240/244, but there was persistent phosphorylation at S235/236 [173]. In accordance with this finding, RSK1 and RSK2 were shown to phosphorylate rpS6 on S235/236 in response to Ras– MAPK-pathway activation, using an mTOR-independent pathway [230]. The RSK mediated S235/236 phosphorylation correlated with formation of the translation pre-initiation complex and increased cap-dependent translation, pointing that RSK provides an additional mitogen- and oncogene-regulated input that links the ERK pathway to the regulation of translation initiation [230]. Translation initiation factor-4B is also phosphorylated by RSK and S6K on S422 [171, 180]. Therefore, phosphorylation and regulation of eIF4B function by RSK and S6K exemplifies the convergence of two major signaling pathways that are involved in translational control. Together, these findings suggest that the mitogenactivated Ras–ERK–RSK signaling module, in parallel with the PI3K–AKT pathway, contains several inputs to stimulate mTORC1 signaling.

Protein Phosphorylation as a Key Mechanism of mTORC1/2 Signaling Pathways 33

It remains unclear, however, how the association of IKKα with mTORC1 can result in the

**Figure 7. mTORC1 crosstalk with the major cytokine signaling pathways.** MAPK pathway impinges on the mTORC1 signaling in a few ways. RSK phosphorylates TSC2 at the C-terminus and, to a lesser extent, the two conserved Akt sites thus inactivating its suppressive effect on mTORC1. RSK also directly impacts the mTORC1 complex by phosphorylation of Raptor and thereby upregulates mTORC1 kinase activity. Besides, RSK phosphorylates the rpS6 and eIF-4B to promote cap-dependent translation in response to Ras–MAPK-pathway activation. ERK1/2 kinase contributes to the activation of mTORC1

association between TSC1 and TSC2. Stress activated signaling pathway might also influence mTORC1 signaling through TSC2 phosphorylation by p38-activated MAPKAPK2 kinase. IKKβ, a major downstream kinase in the TNFα signaling pathway, can associate with and phosphorylate TSC1

signaling through direct phosphorylation of TSC2 and probably through the disruption of the

leading to the inhibition of TSC1/TSC2 and, therefore, the activation of mTORC1.

activation of mTORC1.

## **6.2. mTORC1 and TNFα-IKKβ-TSC1 pathway**

Although the activation of mTORC1 downstream of most cytokines including insulin and growth factors is likely to occur through the Akt and ERK signaling mechanisms described above, accumulating evidence suggests that other cytokines, such as tumor necrosis factor α (TNFα), can also induce mTORC1 activity. TNFα is a proinflammatory cytokine that is involved in many human diseases, including cancer [231, 232]. Early studies implicated the TNFα pathway in mTORC1 activation [233]. Recently, it has been shown that IKKβ (inhibitor of nuclear factor κB (NFκB) kinaseβ; also known as IKBKB), a major downstream kinase in the TNFα signaling pathway, can associate with and phosphorylates TSC1 at S487 and S511, resulting in the inhibition of TSC1–TSC2 and, therefore, the activation of mTORC1 [234]. *Tsc1*−*/*− mouse embryo fibroblasts expressing TSC1 mutants lacking these sites lose their responsiveness to TNF*α* for activation of mTORC1, whereas phosphomimetic mutation lead to a basal increase in mTORC1 signaling. Authors proposed a mechanism involving rapid dissociation of the complex and increased degradation of TSC1. However, the results suggest minimal effects on the stability of the TSC1–TSC2 complex, and the precise mechanism of acute complex inhibition by phosphorylation of these sites is not known. In certain human cancers, TNFα promotes vascular endothelial growth factor (vEGF) expression and angiogenesis through activated mTORC1 signaling as a result of IKKβ mediated suppression of TSC1 [234]. This has provided a plausible mechanism that could link inflammation to cancer pathogenesis. Moreover, TNFα also signals to AKT [231]. Activated AKT in turn induces IKKα (also known as CHUK), [232]. It has been shown that IKKα associates with mTORC1 in an AKT dependent manner [235]. Importantly, IKKα is required for efficient induction of mTORC1 activity by AKT in certain cancer cells [235, 236]. It remains unclear, however, how the association of IKKα with mTORC1 can result in the activation of mTORC1.

32 Protein Phosphorylation in Human Health

contains several inputs to stimulate mTORC1 signaling.

**6.2. mTORC1 and TNFα-IKKβ-TSC1 pathway**

factor-4B (eIF4B) and rpS6 [180]. Although early studies indicated S6K1 and S6K2 as the major rpS6 kinases in somatic cells [228, 229], the role of RSK in regulating site-specific rpS6 phosphorylation and translation in somatic cells has been recently readdressed [230]. Particularly, *in vitro* and *in vivo* evidence suggests that S6Ks phosphorylate every site in rpS6, while RSK predominantly phosphorylates S235 and S236 [230]. Studies from *S6k1*/*S6k2*-knockout mice showed that there was minimal phosphorylation of rpS6 at S240/244, but there was persistent phosphorylation at S235/236 [173]. In accordance with this finding, RSK1 and RSK2 were shown to phosphorylate rpS6 on S235/236 in response to Ras– MAPK-pathway activation, using an mTOR-independent pathway [230]. The RSK mediated S235/236 phosphorylation correlated with formation of the translation pre-initiation complex and increased cap-dependent translation, pointing that RSK provides an additional mitogen- and oncogene-regulated input that links the ERK pathway to the regulation of translation initiation [230]. Translation initiation factor-4B is also phosphorylated by RSK and S6K on S422 [171, 180]. Therefore, phosphorylation and regulation of eIF4B function by RSK and S6K exemplifies the convergence of two major signaling pathways that are involved in translational control. Together, these findings suggest that the mitogenactivated Ras–ERK–RSK signaling module, in parallel with the PI3K–AKT pathway,

Although the activation of mTORC1 downstream of most cytokines including insulin and growth factors is likely to occur through the Akt and ERK signaling mechanisms described above, accumulating evidence suggests that other cytokines, such as tumor necrosis factor α (TNFα), can also induce mTORC1 activity. TNFα is a proinflammatory cytokine that is involved in many human diseases, including cancer [231, 232]. Early studies implicated the TNFα pathway in mTORC1 activation [233]. Recently, it has been shown that IKKβ (inhibitor of nuclear factor κB (NFκB) kinaseβ; also known as IKBKB), a major downstream kinase in the TNFα signaling pathway, can associate with and phosphorylates TSC1 at S487 and S511, resulting in the inhibition of TSC1–TSC2 and, therefore, the activation of mTORC1 [234]. *Tsc1*−*/*− mouse embryo fibroblasts expressing TSC1 mutants lacking these sites lose their responsiveness to TNF*α* for activation of mTORC1, whereas phosphomimetic mutation lead to a basal increase in mTORC1 signaling. Authors proposed a mechanism involving rapid dissociation of the complex and increased degradation of TSC1. However, the results suggest minimal effects on the stability of the TSC1–TSC2 complex, and the precise mechanism of acute complex inhibition by phosphorylation of these sites is not known. In certain human cancers, TNFα promotes vascular endothelial growth factor (vEGF) expression and angiogenesis through activated mTORC1 signaling as a result of IKKβ mediated suppression of TSC1 [234]. This has provided a plausible mechanism that could link inflammation to cancer pathogenesis. Moreover, TNFα also signals to AKT [231]. Activated AKT in turn induces IKKα (also known as CHUK), [232]. It has been shown that IKKα associates with mTORC1 in an AKT dependent manner [235]. Importantly, IKKα is required for efficient induction of mTORC1 activity by AKT in certain cancer cells [235, 236].

**Figure 7. mTORC1 crosstalk with the major cytokine signaling pathways.** MAPK pathway impinges on the mTORC1 signaling in a few ways. RSK phosphorylates TSC2 at the C-terminus and, to a lesser extent, the two conserved Akt sites thus inactivating its suppressive effect on mTORC1. RSK also directly impacts the mTORC1 complex by phosphorylation of Raptor and thereby upregulates mTORC1 kinase activity. Besides, RSK phosphorylates the rpS6 and eIF-4B to promote cap-dependent translation in response to Ras–MAPK-pathway activation. ERK1/2 kinase contributes to the activation of mTORC1 signaling through direct phosphorylation of TSC2 and probably through the disruption of the association between TSC1 and TSC2. Stress activated signaling pathway might also influence mTORC1 signaling through TSC2 phosphorylation by p38-activated MAPKAPK2 kinase. IKKβ, a major downstream kinase in the TNFα signaling pathway, can associate with and phosphorylate TSC1 leading to the inhibition of TSC1/TSC2 and, therefore, the activation of mTORC1.

## **7. Conclusion**

Maintenance of cellular energy homeostasis and life-sustaining activity requires their appropriately adaptation to the continually varying surrounding environment. This adaptation is provided by the differential expression of genes that is strictly controlled at the levels of transcription and translation. To provide the rapid response to the environmental cues cells switch vast number of intracellular signaling cascades that define the activity of key proteins responsible for transcription and translation regulation. This implies operative and directed changes in the activities of proteins mediating the signal transduction. Phosphorylation represents one of the most important intracellular regulatory molecular mechanisms since it provides the rapid and reversible activation or downregulation of protein activities. Not surprisingly, mTOR signaling network, which integrates and promotes the prompt respond to environmental changes is mainly regulated through this type of posttranslational modification. mTOR is known as a "switch master" that converts vast array of nutrient-, cytokine-, energy- and stress-sensitive signals into the alterations of cellular metabolism including protein and lipid biosynthesis and autophagy. Indeed such resources consuming processes as growth and proliferation could occur only under the conditions of nutrient and energy sufficiency. When energy or amino acids become limiting, cell growth needs to be restricted and protein production needs to be downregulated so that cells can use their limited resources to survive. mTORC1 contributes to overall capdependent translation including initiation and elongation steps by several different pathways. Significantly, all of these pathways use phosphorylation as common molecular mechanism of regulation. Most of the proteins from mTOR-dependent pathways (for instance TSC2, Akt, S6K, etc.) contain multiple phosphorylating sites, which mediate stimulating or negative effect on their activity. Some of mTOR partners are characterized by the hierarchical mode of phosphorylation (rpS6, Akt as the examples), whereby each previous phosphorylation opens the opportunity for the subsequent ones. Interestingly, one certain site could serve as phosphorylation target for more than one kinase, therefore implementing the competitive mechanism of regulation (for instance, eIF4B S422). The complexity of mTOR signaling increases due to the presence of positive or negative feedback loops as well as crosstalk with other pathways. The number of reported phosphorylation sites throughout mTOR pathways constantly increases although the precise molecular meaning of several already discovered phosphorylation events remains unclear. This is also true to some molecular mechanisms of mTORC1 and especially mTORC2 functioning. About mTORC1 signaling, a number of issues remain unresolved. For example, aside from S6K, 4EBP1 and ULK1, the downstream direct targets that mediate the cellular effects of TORC1 signaling are largely unaccounted for. In addition, how the specificity of TORC1 signaling is achieved and how multiple signals are integrated is not known. Concerning TORC2, the upstream regulators are poorly defined. This knowledge seems to be of great significance since mTOR is considered a central node of intracellular signaling network and deregulation of its activity strongly contributes to the wide spectrum of human diseases. Further studies will give us a better understanding of the whole picture of mTORC1/mTORC2 functioning that could be applied to the development of new approaches to the treatment of mTOR-associated diseases.

Protein Phosphorylation as a Key Mechanism of mTORC1/2 Signaling Pathways 35

*Oncogenes Regulation Department, N.N. Blokhin Russian Cancer Research Center, Moscow, Russia*

*Oncogenes Regulation Department, N.N. Blokhin Russian Cancer Research Center, Moscow, Russia*

[1] Dennis PB, Jaeschke A, Saitoh M, Fowler B, Kozma SC, et al. (2001) Mammalian TOR: a

[2] Inoki K, Zhu T, Guan KL. (2003) TSC2 mediates cellular energy response to control cell

[3] Jacinto E, Loewith R, Schmidt A, Lin S, Ruegg MA, et al. (2004) Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nature cell

[4] Sarbassov DD, Ali SM, Kim DH, Guertin DA, Latek RR, et al. (2004) Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Current biology: CB.14(14):1296-302. Epub

[5] Loewith R, Jacinto E, Wullschleger S, Lorberg A, Crespo JL, et al. (2002) Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth

[6] Vezina C, Kudelski A, Sehgal SN. (1975) Rapamycin (AY-22,989), a new antifungal antibiotic. I. Taxonomy of the producing streptomycete and isolation of the active

[7] Eng CP, Sehgal SN, Vezina C. (1984) Activity of rapamycin (AY-22,989) against transplanted tumors. The Journal of antibiotics.37(10):1231-7. Epub 1984/10/01. [8] Douros J, Suffness M. (1981) New antitumor substances of natural origin. Cancer

[9] Sehgal SN, Baker H, Vezina C. (1975) Rapamycin (AY-22,989), a new antifungal antibiotic. II. Fermentation, isolation and characterization. The Journal of

[10] Cafferkey R, Young PR, McLaughlin MM, Bergsma DJ, Koltin Y, et al. (1993) Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity. Mol Cell Biol.13(10):6012-23. Epub

[11] Heitman J, Movva NR, Hall MN. (1991) Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science.253(5022):905-9. Epub 1991/08/23. [12] Chen J, Zheng XF, Brown EJ, Schreiber SL. (1995) Identification of an 11-kDa FKBP12 rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proceedings of the National Academy

of Sciences of the United States of America.92(11):4947-51. Epub 1995/05/23.

homeostatic ATP sensor. Science.294(5544):1102-5. Epub 2001/11/03.

growth and survival. Cell.115(5):577-90. Epub 2003/12/04.

control. Molecular cell.10(3):457-68. Epub 2002/11/01.

treatment reviews.8(1):63-87. Epub 1981/03/01.

antibiotics.28(10):727-32. Epub 1975/10/01.

principle. The Journal of antibiotics.28(10):721-6. Epub 1975/10/01.

biology.6(11):1122-8. Epub 2004/10/07.

**Author details**

Elena Tchevkina *Corresponding Author*

Andrey Komelkov

**8. References**

2004/07/23.

1993/10/01.

## **Author details**

34 Protein Phosphorylation in Human Health

approaches to the treatment of mTOR-associated diseases.

Maintenance of cellular energy homeostasis and life-sustaining activity requires their appropriately adaptation to the continually varying surrounding environment. This adaptation is provided by the differential expression of genes that is strictly controlled at the levels of transcription and translation. To provide the rapid response to the environmental cues cells switch vast number of intracellular signaling cascades that define the activity of key proteins responsible for transcription and translation regulation. This implies operative and directed changes in the activities of proteins mediating the signal transduction. Phosphorylation represents one of the most important intracellular regulatory molecular mechanisms since it provides the rapid and reversible activation or downregulation of protein activities. Not surprisingly, mTOR signaling network, which integrates and promotes the prompt respond to environmental changes is mainly regulated through this type of posttranslational modification. mTOR is known as a "switch master" that converts vast array of nutrient-, cytokine-, energy- and stress-sensitive signals into the alterations of cellular metabolism including protein and lipid biosynthesis and autophagy. Indeed such resources consuming processes as growth and proliferation could occur only under the conditions of nutrient and energy sufficiency. When energy or amino acids become limiting, cell growth needs to be restricted and protein production needs to be downregulated so that cells can use their limited resources to survive. mTORC1 contributes to overall capdependent translation including initiation and elongation steps by several different pathways. Significantly, all of these pathways use phosphorylation as common molecular mechanism of regulation. Most of the proteins from mTOR-dependent pathways (for instance TSC2, Akt, S6K, etc.) contain multiple phosphorylating sites, which mediate stimulating or negative effect on their activity. Some of mTOR partners are characterized by the hierarchical mode of phosphorylation (rpS6, Akt as the examples), whereby each previous phosphorylation opens the opportunity for the subsequent ones. Interestingly, one certain site could serve as phosphorylation target for more than one kinase, therefore implementing the competitive mechanism of regulation (for instance, eIF4B S422). The complexity of mTOR signaling increases due to the presence of positive or negative feedback loops as well as crosstalk with other pathways. The number of reported phosphorylation sites throughout mTOR pathways constantly increases although the precise molecular meaning of several already discovered phosphorylation events remains unclear. This is also true to some molecular mechanisms of mTORC1 and especially mTORC2 functioning. About mTORC1 signaling, a number of issues remain unresolved. For example, aside from S6K, 4EBP1 and ULK1, the downstream direct targets that mediate the cellular effects of TORC1 signaling are largely unaccounted for. In addition, how the specificity of TORC1 signaling is achieved and how multiple signals are integrated is not known. Concerning TORC2, the upstream regulators are poorly defined. This knowledge seems to be of great significance since mTOR is considered a central node of intracellular signaling network and deregulation of its activity strongly contributes to the wide spectrum of human diseases. Further studies will give us a better understanding of the whole picture of mTORC1/mTORC2 functioning that could be applied to the development of new

**7. Conclusion**

Elena Tchevkina *Corresponding Author Oncogenes Regulation Department, N.N. Blokhin Russian Cancer Research Center, Moscow, Russia*

Andrey Komelkov

*Oncogenes Regulation Department, N.N. Blokhin Russian Cancer Research Center, Moscow, Russia*

## **8. References**

[13] Choi J, Chen J, Schreiber SL, Clardy J. (1996) Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP. Science.273(5272):239- 42. Epub 1996/07/12.

Protein Phosphorylation as a Key Mechanism of mTORC1/2 Signaling Pathways 37

[29] Soliman GA, Acosta-Jaquez HA, Dunlop EA, Ekim B, Maj NE, et al. (2010) mTOR Ser-2481 autophosphorylation monitors mTORC-specific catalytic activity and clarifies rapamycin mechanism of action. The Journal of biological chemistry.285(11):7866-79.

[30] Caron E, Ghosh S, Matsuoka Y, Ashton-Beaucage D, Therrien M, et al. (2010) A comprehensive map of the mTOR signaling network. Molecular systems biology.6:453.

[31] Acosta-Jaquez HA, Keller JA, Foster KG, Ekim B, Soliman GA, et al. (2009) Site-specific mTOR phosphorylation promotes mTORC1-mediated signaling and cell growth. Mol

[32] Chiang GG, Abraham RT. (2005) Phosphorylation of mammalian target of rapamycin (mTOR) at Ser-2448 is mediated by p70S6 kinase. The Journal of biological

[33] Peterson RT, Beal PA, Comb MJ, Schreiber SL. (2000) FKBP12-rapamycin-associated protein (FRAP) autophosphorylates at serine 2481 under translationally repressive

[35] Russell RC, Fang C, Guan KL. (2011) An emerging role for TOR signaling in mammalian tissue and stem cell physiology. Development.138(16):3343-56. Epub

[36] Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, et al. (2010) Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by

[37] Boulbes DR, Shaiken T, Sarbassov dos D. (2011) Endoplasmic reticulum is a main localization site of mTORC2. Biochemical and biophysical research

[38] Zinzalla V, Stracka D, Oppliger W, Hall MN. (2011) Activation of mTORC2 by

[39] Kim JE, Chen J. (2000) Cytoplasmic-nuclear shuttling of FKBP12-rapamycin-associated protein is involved in rapamycin-sensitive signaling and translation initiation. Proceedings of the National Academy of Sciences of the United States of

[40] Yip CK, Murata K, Walz T, Sabatini DM, Kang SA. (2010) Structure of the human mTOR complex I and its implications for rapamycin inhibition. Molecular cell.38(5):768-

[41] Peterson TR, Laplante M, Thoreen CC, Sancak Y, Kang SA, et al. (2009) DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for

[42] Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, et al. (2002) mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery.

[43] Hara K, Maruki Y, Long X, Yoshino K, Oshiro N, et al. (2002) Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell.110(2):177-89. Epub 2002/08/02.

conditions. The Journal of biological chemistry.275(10):7416-23. Epub 2000/03/04. [34] Edinger AL, Linardic CM, Chiang GG, Thompson CB, Abraham RT. (2003) Differential effects of rapamycin on mammalian target of rapamycin signaling functions in

mammalian cells. Cancer research.63(23):8451-60. Epub 2003/12/18.

association with the ribosome. Cell.144(5):757-68. Epub 2011/03/08.

Epub 2009/12/22.

Epub 2010/12/24.

2011/07/28.

Cell Biol.29(15):4308-24. Epub 2009/06/03.

chemistry.280(27):25485-90. Epub 2005/05/19.

amino acids. Cell.141(2):290-303. Epub 2010/04/13.

communications.413(1):46-52. Epub 2011/08/27.

America.97(26):14340-5. Epub 2000/12/13.

Cell.110(2):163-75. Epub 2002/08/02.

their survival. Cell.137(5):873-86. Epub 2009/05/19.

74. Epub 2010/06/15.

[29] Soliman GA, Acosta-Jaquez HA, Dunlop EA, Ekim B, Maj NE, et al. (2010) mTOR Ser-2481 autophosphorylation monitors mTORC-specific catalytic activity and clarifies rapamycin mechanism of action. The Journal of biological chemistry.285(11):7866-79. Epub 2009/12/22.

36 Protein Phosphorylation in Human Health

42. Epub 1996/07/12.

Epub 1994/06/30.

[13] Choi J, Chen J, Schreiber SL, Clardy J. (1996) Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP. Science.273(5272):239-

[14] Sabers CJ, Martin MM, Brunn GJ, Williams JM, Dumont FJ, et al. (1995) Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells. The Journal of

[15] Brown EJ, Albers MW, Shin TB, Ichikawa K, Keith CT, et al. (1994) A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature.369(6483):756-8.

[16] Chiu MI, Katz H, Berlin V. (1994) RAPT1, a mammalian homolog of yeast Tor, interacts with the FKBP12/rapamycin complex. Proceedings of the National Academy of Sciences

[17] Sabatini DM, Erdjument-Bromage H, Lui M, Tempst P, Snyder SH. (1994) RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is

[18] Chen Y, Chen H, Rhoad AE, Warner L, Caggiano TJ, et al. (1994) A putative sirolimus (rapamycin) effector protein. Biochemical and biophysical research

[19] Keith CT, Schreiber SL. (1995) PIK-related kinases: DNA repair, recombination, and cell

[20] Kunz J, Henriquez R, Schneider U, Deuter-Reinhard M, Movva NR, et al. (1993) Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog

[21] Wiederrecht GJ, Sabers CJ, Brunn GJ, Martin MM, Dumont FJ, et al. (1995) Mechanism of action of rapamycin: new insights into the regulation of G1-phase progression in

[22] Hay N, Sonenberg N. (2004) Upstream and downstream of mTOR. Genes &

[23] Janus A, Robak T, Smolewski P. (2005) The mammalian target of the rapamycin (mTOR) kinase pathway: its role in tumourigenesis and targeted antitumour therapy.

[24] Gangloff YG, Mueller M, Dann SG, Svoboda P, Sticker M, et al. (2004) Disruption of the mouse mTOR gene leads to early postimplantation lethality and prohibits embryonic

[25] Murakami M, Ichisaka T, Maeda M, Oshiro N, Hara K, et al. (2004) mTOR is essential for growth and proliferation in early mouse embryos and embryonic stem cells. Mol

[26] Gingras AC, Raught B, Sonenberg N. (2001) Regulation of translation initiation by

[27] Perry J, Kleckner N. (2003) The ATRs, ATMs, and TORs are giant HEAT repeat proteins.

[28] Foster KG, Fingar DC. (2010) Mammalian target of rapamycin (mTOR): conducting the cellular signaling symphony. The Journal of biological chemistry.285(19):14071-7. Epub

biological chemistry.270(2):815-22. Epub 1995/01/13.

of the United States of America.91(26):12574-8. Epub 1994/12/20.

homologous to yeast TORs. Cell.78(1):35-43. Epub 1994/07/15.

cycle checkpoints. Science.270(5233):50-1. Epub 1995/10/06.

required for G1 progression. Cell.73(3):585-96. Epub 1993/05/07.

eukaryotic cells. Progress in cell cycle research.1:53-71. Epub 1995/01/01.

Cellular & molecular biology letters.10(3):479-98. Epub 2005/10/12.

stem cell development. Mol Cell Biol.24(21):9508-16. Epub 2004/10/16.

FRAP/mTOR. Genes & development.15(7):807-26. Epub 2001/04/12.

communications.203(1):1-7. Epub 1994/08/30.

development.18(16):1926-45. Epub 2004/08/18.

Cell Biol.24(15):6710-8. Epub 2004/07/16.

Cell.112(2):151-5. Epub 2003/01/30.

2010/03/17.

[44] Zhou H, Huang S. (2010) The complexes of mammalian target of rapamycin. Current protein & peptide science.11(6):409-24. Epub 2010/05/25.

Protein Phosphorylation as a Key Mechanism of mTORC1/2 Signaling Pathways 39

[58] Jacinto E, Facchinetti V, Liu D, Soto N, Wei S, et al. (2006) SIN1/MIP1 maintains rictormTOR complex integrity and regulates Akt phosphorylation and substrate specificity.

[59] Yang Q, Inoki K, Ikenoue T, Guan KL. (2006) Identification of Sin1 as an essential TORC2 component required for complex formation and kinase activity. Genes &

[60] Wilkinson MG, Pino TS, Tournier S, Buck V, Martin H, et al. (1999) Sin1: an evolutionarily conserved component of the eukaryotic SAPK pathway. The EMBO

[61] Schroder WA, Buck M, Cloonan N, Hancock JF, Suhrbier A, et al. (2007) Human Sin1 contains Ras-binding and pleckstrin homology domains and suppresses Ras signalling.

[62] Yang Q, Guan KL. (2007) Expanding mTOR signaling. Cell research.17(8):666-81. Epub

[63] Thedieck K, Polak P, Kim ML, Molle KD, Cohen A, et al. (2007) PRAS40 and PRR5-like protein are new mTOR interactors that regulate apoptosis. PloS one.2(11):e1217. Epub

[64] Tee AR, Fingar DC, Manning BD, Kwiatkowski DJ, Cantley LC, et al. (2002) Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling. Proceedings of the National Academy of Sciences of the United States of America.99(21):13571-6. Epub 2002/09/25. [65] Manning BD, Cantley LC. (2003) United at last: the tuberous sclerosis complex gene products connect the phosphoinositide 3-kinase/Akt pathway to mammalian target of rapamycin (mTOR) signalling. Biochemical Society transactions.31(Pt 3):573-8. Epub

[66] Kandt RS, Haines JL, Smith M, Northrup H, Gardner RJ, et al. (1992) Linkage of an important gene locus for tuberous sclerosis to a chromosome 16 marker for polycystic

[67] Consortium ECTS. (1993) Identification and characterization of the tuberous sclerosis

[68] van Slegtenhorst M, de Hoogt R, Hermans C, Nellist M, Janssen B, et al. (1997) Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34.

[69] Hodges AK, Li S, Maynard J, Parry L, Braverman R, et al. (2001) Pathological mutations in TSC1 and TSC2 disrupt the interaction between hamartin and tuberin. Human

[70] Garami A, Zwartkruis FJ, Nobukuni T, Joaquin M, Roccio M, et al. (2003) Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and

[71] Benvenuto G, Li S, Brown SJ, Braverman R, Vass WC, et al. (2000) The tuberous sclerosis-1 (TSC1) gene product hamartin suppresses cell growth and augments the expression of the TSC2 product tuberin by inhibiting its ubiquitination.

kidney disease. Nature genetics.2(1):37-41. Epub 1992/09/01.

gene on chromosome 16. Cell.75(7):1305-15. Epub 1993/12/31.

Science.277(5327):805-8. Epub 1997/08/08.

molecular genetics.10(25):2899-905. Epub 2001/12/14.

2. Molecular cell.11(6):1457-66. Epub 2003/06/25.

Oncogene.19(54):6306-16. Epub 2001/02/15.

Cell.127(1):125-37. Epub 2006/09/12.

development.20(20):2820-32. Epub 2006/10/18.

Cellular signalling.19(6):1279-89. Epub 2007/02/17.

journal.18(15):4210-21. Epub 1999/08/03.

2007/08/08.

2007/11/22.

2003/05/30.

[58] Jacinto E, Facchinetti V, Liu D, Soto N, Wei S, et al. (2006) SIN1/MIP1 maintains rictormTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell.127(1):125-37. Epub 2006/09/12.

38 Protein Phosphorylation in Human Health

Epub 2008/04/29.

Epub 2007/03/28.

2007/05/23.

2008/03/29.

[44] Zhou H, Huang S. (2010) The complexes of mammalian target of rapamycin. Current

[45] Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, et al. (2008) AMPK phosphorylation of raptor mediates a metabolic checkpoint. Molecular cell.30(2):214-26.

[46] Carriere A, Cargnello M, Julien LA, Gao H, Bonneil E, et al. (2008) Oncogenic MAPK signaling stimulates mTORC1 activity by promoting RSK-mediated raptor

[47] Wang L, Lawrence JC, Jr., Sturgill TW, Harris TE. (2009) Mammalian target of rapamycin complex 1 (mTORC1) activity is associated with phosphorylation of raptor

[49] Kovacina KS, Park GY, Bae SS, Guzzetta AW, Schaefer E, et al. (2003) Identification of a proline-rich Akt substrate as a 14-3-3 binding partner. The Journal of biological

[50] Oshiro N, Takahashi R, Yoshino K, Tanimura K, Nakashima A, et al. (2007) The prolinerich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of mammalian target of rapamycin complex 1. The Journal of biological chemistry.282(28):20329-39. Epub

[51] Wang L, Harris TE, Lawrence JC, Jr. (2008) Regulation of proline-rich Akt substrate of 40 kDa (PRAS40) function by mammalian target of rapamycin complex 1 (mTORC1) mediated phosphorylation. The Journal of biological chemistry.283(23):15619-27. Epub

[52] Kim DH, Sarbassov DD, Ali SM, Latek RR, Guntur KV, et al. (2003) GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR. Molecular cell.11(4):895-904. Epub 2003/04/30. [53] Frias MA, Thoreen CC, Jaffe JD, Schroder W, Sculley T, et al. (2006) mSin1 is necessary for Akt/PKB phosphorylation, and its isoforms define three distinct mTORC2s. Current

[54] Pearce LR, Huang X, Boudeau J, Pawlowski R, Wullschleger S, et al. (2007) Identification of Protor as a novel Rictor-binding component of mTOR complex-2. The

[55] Woo SY, Kim DH, Jun CB, Kim YM, Haar EV, et al. (2007) PRR5, a novel component of mTOR complex 2, regulates platelet-derived growth factor receptor beta expression and

[57] Vander Haar E, Lee SI, Bandhakavi S, Griffin TJ, Kim DH. (2007) Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nature cell biology.9(3):316-23.

signaling. The Journal of biological chemistry.282(35):25604-12. Epub 2007/06/30. [56] Sarbassov DD, Ali SM, Sengupta S, Sheen JH, Hsu PP, et al. (2006) Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Molecular

by mTOR. The Journal of biological chemistry.284(22):14693-7. Epub 2009/04/07. [48] Sancak Y, Thoreen CC, Peterson TR, Lindquist RA, Kang SA, et al. (2007) PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase. Molecular cell.25(6):903-15.

phosphorylation. Current biology : CB.18(17):1269-77. Epub 2008/08/30.

protein & peptide science.11(6):409-24. Epub 2010/05/25.

chemistry.278(12):10189-94. Epub 2003/01/14.

biology : CB.16(18):1865-70. Epub 2006/08/22.

cell.22(2):159-68. Epub 2006/04/11.

Epub 2007/02/06.

Biochemical journal.405(3):513-22. Epub 2007/04/28.

[72] Chong-Kopera H, Inoki K, Li Y, Zhu T, Garcia-Gonzalo FR, et al. (2006) TSC1 stabilizes TSC2 by inhibiting the interaction between TSC2 and the HERC1 ubiquitin ligase. The Journal of biological chemistry.281(13):8313-6. Epub 2006/02/09.

Protein Phosphorylation as a Key Mechanism of mTORC1/2 Signaling Pathways 41

phosphorylation of tuberin. The Journal of biological chemistry.277(38):35364-70. Epub

[89] Ballif BA, Roux PP, Gerber SA, MacKeigan JP, Blenis J, et al. (2005) Quantitative phosphorylation profiling of the ERK/p90 ribosomal S6 kinase-signaling cassette and its targets, the tuberous sclerosis tumor suppressors. Proceedings of the National Academy

[90] Gao X, Zhang Y, Arrazola P, Hino O, Kobayashi T, et al. (2002) Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling. Nature cell biology.4(9):699-704. Epub

[91] Tee AR, Proud CG. (2002) Caspase cleavage of initiation factor 4E-binding protein 1 yields a dominant inhibitor of cap-dependent translation and reveals a novel regulatory

[92] Huang J, Manning BD. (2008) The TSC1-TSC2 complex: a molecular switchboard controlling cell growth. The Biochemical journal.412(2):179-90. Epub 2008/05/10. [93] Cai SL, Tee AR, Short JD, Bergeron JM, Kim J, et al. (2006) Activity of TSC2 is inhibited by AKT-mediated phosphorylation and membrane partitioning. The Journal of cell

[94] Nellist M, Goedbloed MA, de Winter C, Verhaaf B, Jankie A, et al. (2002) Identification and characterization of the interaction between tuberin and 14-3-3zeta. The Journal of

[95] Shumway SD, Li Y, Xiong Y. (2003) 14-3-3beta binds to and negatively regulates the tuberous sclerosis complex 2 (TSC2) tumor suppressor gene product, tuberin. The

[96] Dong J, Pan D. (2004) Tsc2 is not a critical target of Akt during normal Drosophila

[97] Wang YH, Huang ML. (2009) Reduction of Lobe leads to TORC1 hypoactivation that induces ectopic Jak/STAT signaling to impair Drosophila eye development.

[98] Jacinto E, Hall MN. (2003) Tor signalling in bugs, brain and brawn. Nature reviews

[99] Long X, Ortiz-Vega S, Lin Y, Avruch J. (2005) Rheb binding to mammalian target of rapamycin (mTOR) is regulated by amino acid sufficiency. The Journal of biological

[100] Smith EM, Finn SG, Tee AR, Browne GJ, Proud CG. (2005) The tuberous sclerosis protein TSC2 is not required for the regulation of the mammalian target of rapamycin by amino acids and certain cellular stresses. The Journal of biological

[101] Wang X, Proud CG. (2009) Nutrient control of TORC1, a cell-cycle regulator. Trends in

[102] Roccio M, Bos JL, Zwartkruis FJ. (2006) Regulation of the small GTPase Rheb by amino

[103] Byfield MP, Murray JT, Backer JM. (2005) hVps34 is a nutrient-regulated lipid kinase required for activation of p70 S6 kinase. The Journal of biological

of Sciences of the United States of America.102(3):667-72. Epub 2005/01/14.

motif. Mol Cell Biol.22(6):1674-83. Epub 2002/02/28.

biological chemistry.277(42):39417-24. Epub 2002/08/15.

Journal of biological chemistry.278(4):2089-92. Epub 2002/12/07.

Mechanisms of development.126(10):781-90. Epub 2009/09/08.

Molecular cell biology.4(2):117-26. Epub 2003/02/04.

chemistry.280(25):23433-6. Epub 2005/05/10.

chemistry.280(19):18717-27. Epub 2005/03/18.

acids. Oncogene.25(5):657-64. Epub 2005/09/20.

chemistry.280(38):33076-82. Epub 2005/07/29.

cell biology.19(6):260-7. Epub 2009/05/08.

development. Genes & development.18(20):2479-84. Epub 2004/10/07.

biology.173(2):279-89. Epub 2006/04/26.

2002/08/09.

2002/08/13.

phosphorylation of tuberin. The Journal of biological chemistry.277(38):35364-70. Epub 2002/08/09.

[89] Ballif BA, Roux PP, Gerber SA, MacKeigan JP, Blenis J, et al. (2005) Quantitative phosphorylation profiling of the ERK/p90 ribosomal S6 kinase-signaling cassette and its targets, the tuberous sclerosis tumor suppressors. Proceedings of the National Academy of Sciences of the United States of America.102(3):667-72. Epub 2005/01/14.

40 Protein Phosphorylation in Human Health

2007/11/10.

Epub 1997/01/31.

Epub 2005/09/20.

2005/02/19.

[72] Chong-Kopera H, Inoki K, Li Y, Zhu T, Garcia-Gonzalo FR, et al. (2006) TSC1 stabilizes TSC2 by inhibiting the interaction between TSC2 and the HERC1 ubiquitin ligase. The

[73] Tee AR, Blenis J, Proud CG. (2005) Analysis of mTOR signaling by the small G-proteins,

[74] Long X, Lin Y, Ortiz-Vega S, Yonezawa K, Avruch J. (2005) Rheb binds and regulates

[75] Bai X, Ma D, Liu A, Shen X, Wang QJ, et al. (2007) Rheb activates mTOR by antagonizing its endogenous inhibitor, FKBP38. Science.318(5852):977-80. Epub

[76] Rosner M, Hofer K, Kubista M, Hengstschlager M. (2003) Cell size regulation by the human TSC tumor suppressor proteins depends on PI3K and FKBP38.

[77] Manning BD, Cantley LC. (2007) AKT/PKB signaling: navigating downstream.

[78] Zhao L, Vogt PK. (2010) Hot-spot mutations in p110alpha of phosphatidylinositol 3 kinase (pI3K): differential interactions with the regulatory subunit p85 and with RAS.

[79] Franke TF, Kaplan DR, Cantley LC, Toker A. (1997) Direct regulation of the Akt protooncogene product by phosphatidylinositol-3,4-bisphosphate. Science.275(5300):665-8.

[80] Martelli AM, Evangelisti C, Chiarini F, Grimaldi C, Cappellini A, et al. (2010) The emerging role of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling network in normal myelopoiesis and leukemogenesis. Biochimica et

[81] McManus EJ, Collins BJ, Ashby PR, Prescott AR, Murray-Tait V, et al. (2004) The in vivo role of PtdIns(3,4,5)P3 binding to PDK1 PH domain defined by knockin mutation. The

[82] Hay N. (2005) The Akt-mTOR tango and its relevance to cancer. Cancer Cell.8(3):179-83.

[83] Toker A, Newton AC. (2000) Cellular signaling: pivoting around PDK-1.

[84] Guertin DA, Stevens DM, Thoreen CC, Burds AA, Kalaany NY, et al. (2006) Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1. Developmental

[85] Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. (2005) Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science.307(5712):1098-101. Epub

[86] Potter CJ, Pedraza LG, Xu T. (2002) Akt regulates growth by directly phosphorylating

[87] Inoki K, Li Y, Zhu T, Wu J, Guan KL. (2002) TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nature cell biology.4(9):648-57. Epub 2002/08/13. [88] Dan HC, Sun M, Yang L, Feldman RI, Sui XM, et al. (2002) Phosphatidylinositol 3 kinase/Akt pathway regulates tuberous sclerosis tumor suppressor complex by

Journal of biological chemistry.281(13):8313-6. Epub 2006/02/09.

Rheb and RhebL1. FEBS letters.579(21):4763-8. Epub 2005/08/16.

Oncogene.22(31):4786-98. Epub 2003/08/02.

Cell.129(7):1261-74. Epub 2007/07/03.

Cell Cycle.9(3):596-600. Epub 2009/12/17.

biophysica acta.1803(9):991-1002. Epub 2010/04/20.

EMBO journal.23(10):2071-82. Epub 2004/04/30.

Tsc2. Nature cell biology.4(9):658-65. Epub 2002/08/13.

Cell.103(2):185-8. Epub 2000/11/01.

cell.11(6):859-71. Epub 2006/12/05.

the mTOR kinase. Current biology : CB.15(8):702-13. Epub 2005/04/28.

	- [104] Nobukuni T, Joaquin M, Roccio M, Dann SG, Kim SY, et al. (2005) Amino acids mediate mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase. Proceedings of the National Academy of Sciences of the United States of America.102(40):14238-43. Epub 2005/09/24.

Protein Phosphorylation as a Key Mechanism of mTORC1/2 Signaling Pathways 43

rapamycin-p70 S6 kinase 1 pathway. The Journal of biological chemistry.278(46):45117-

[120] Chen Y, Rodrik V, Foster DA. (2005) Alternative phospholipase D/mTOR survival

[121] Hui L, Abbas T, Pielak RM, Joseph T, Bargonetti J, et al. (2004) Phospholipase D elevates the level of MDM2 and suppresses DNA damage-induced increases in p53.

[122] Ohguchi K, Banno Y, Nakagawa Y, Akao Y, Nozawa Y. (2005) Negative regulation of melanogenesis by phospholipase D1 through mTOR/p70 S6 kinase 1 signaling in mouse B16 melanoma cells. Journal of cellular physiology.205(3):444-51. Epub 2005/05/17. [123] Fukami K, Takenawa T. (1992) Phosphatidic acid that accumulates in platelet-derived growth factor-stimulated Balb/c 3T3 cells is a potential mitogenic signal. The Journal of

[124] Sun Y, Fang Y, Yoon MS, Zhang C, Roccio M, et al. (2008) Phospholipase D1 is an effector of Rheb in the mTOR pathway. Proceedings of the National Academy of

[125] Ha SH, Kim DH, Kim IS, Kim JH, Lee MN, et al. (2006) PLD2 forms a functional complex with mTOR/raptor to transduce mitogenic signals. Cellular

[126] Toschi A, Lee E, Xu L, Garcia A, Gadir N, et al. (2009) Regulation of mTORC1 and mTORC2 complex assembly by phosphatidic acid: competition with rapamycin. Mol

[127] Xu L, Salloum D, Medlin PS, Saqcena M, Yellen P, et al. (2011) Phospholipase D mediates nutrient input to mammalian target of rapamycin complex 1 (mTORC1). The

[128] Frohman MA, Morris AJ. (1999) Phospholipase D structure and regulation. Chemistry

[129] Knizhnik AV, Kovaleva OV, Komelkov AV, Trukhanova LS, Rybko VA, et al. (2012) Arf6 promotes cell proliferation via the PLD-mTORC1 and p38MAPK pathways.

[130] Hardie DG. (2007) AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy. Nature reviews Molecular cell biology.8(10):774-85. Epub 2007/08/23. [131] Kahn BB, Alquier T, Carling D, Hardie DG. (2005) AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell

[132] Shaw RJ. (2009) LKB1 and AMP-activated protein kinase control of mTOR signalling

[133] Sanders MJ, Grondin PO, Hegarty BD, Snowden MA, Carling D. (2007) Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade. The

[134] Oakhill JS, Steel R, Chen ZP, Scott JW, Ling N, et al. (2011) AMPK is a direct adenylate

[135] Xiao B, Sanders MJ, Underwood E, Heath R, Mayer FV, et al. (2011) Structure of mammalian AMPK and its regulation by ADP. Nature.472(7342):230-3. Epub

charge-regulated protein kinase. Science.332(6036):1433-5. Epub 2011/06/18.

Sciences of the United States of America.105(24):8286-91. Epub 2008/06/14.

Journal of biological chemistry.286(29):25477-86. Epub 2011/05/31.

Journal of cellular biochemistry.113(1):360-71. Epub 2011/09/20.

and growth. Acta Physiol (Oxf).196(1):65-80. Epub 2009/02/28.

Biochemical journal.403(1):139-48. Epub 2006/12/07.

and physics of lipids.98(1-2):127-40. Epub 1999/06/08.

signal in human breast cancer cells. Oncogene.24(4):672-9. Epub 2004/12/08.

27. Epub 2003/09/10.

Mol Cell Biol.24(13):5677-86. Epub 2004/06/17.

signalling.18(12):2283-91. Epub 2006/07/14.

Cell Biol.29(6):1411-20. Epub 2008/12/31.

metabolism.1(1):15-25. Epub 2005/08/02.

2011/03/15.

biological chemistry.267(16):10988-93. Epub 1992/06/05.

rapamycin-p70 S6 kinase 1 pathway. The Journal of biological chemistry.278(46):45117- 27. Epub 2003/09/10.

[120] Chen Y, Rodrik V, Foster DA. (2005) Alternative phospholipase D/mTOR survival signal in human breast cancer cells. Oncogene.24(4):672-9. Epub 2004/12/08.

42 Protein Phosphorylation in Human Health

2010/03/17.

65. Epub 2008/05/08.

4. Epub 2007/01/11.

Epub 2002/06/28.

2001/12/01.

America.102(40):14238-43. Epub 2005/09/24.

of cell biology.181(4):655-66. Epub 2008/05/14.

The Biochemical journal.410(1):1-17. Epub 2008/01/25.

Science.320(5882):1496-501. Epub 2008/05/24.

States of America.103(12):4741-6. Epub 2006/03/16.

journal.403(1):13-20. Epub 2007/01/27.

[104] Nobukuni T, Joaquin M, Roccio M, Dann SG, Kim SY, et al. (2005) Amino acids mediate mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase. Proceedings of the National Academy of Sciences of the United States of

[105] Findlay GM, Yan L, Procter J, Mieulet V, Lamb RF. (2007) A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling. The Biochemical

[106] Juhasz G, Hill JH, Yan Y, Sass M, Baehrecke EH, et al. (2008) The class III PI(3)K Vps34 promotes autophagy and endocytosis but not TOR signaling in Drosophila. The Journal

[107] Yan L, Mieulet V, Burgess D, Findlay GM, Sully K, et al. (2010) PP2A T61 epsilon is an inhibitor of MAP4K3 in nutrient signaling to mTOR. Molecular cell.37(5):633-42. Epub

[108] Gulati P, Gaspers LD, Dann SG, Joaquin M, Nobukuni T, et al. (2008) Amino acids activate mTOR complex 1 via Ca2+/CaM signaling to hVps34. Cell metabolism.7(5):456-

[109] Backer JM. (2008) The regulation and function of Class III PI3Ks: novel roles for Vps34.

[110] Tassa A, Roux MP, Attaix D, Bechet DM. (2003) Class III phosphoinositide 3-kinase-- Beclin1 complex mediates the amino acid-dependent regulation of autophagy in C2C12

[111] Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, et al. (2008) The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1.

[112] Kim E, Goraksha-Hicks P, Li L, Neufeld TP, Guan KL. (2008) Regulation of TORC1 by Rag GTPases in nutrient response. Nature cell biology.10(8):935-45. Epub 2008/07/08. [113] Exton JH. (2002) Phospholipase D-structure, regulation and function. Reviews of

[114] Foster DA. (2007) Regulation of mTOR by phosphatidic acid? Cancer research.67(1):1-

[115] Fang Y, Vilella-Bach M, Bachmann R, Flanigan A, Chen J. (2001) Phosphatidic acidmediated mitogenic activation of mTOR signaling. Science.294(5548):1942-5. Epub

[116] Hornberger TA, Chu WK, Mak YW, Hsiung JW, Huang SA, et al. (2006) The role of phospholipase D and phosphatidic acid in the mechanical activation of mTOR signaling in skeletal muscle. Proceedings of the National Academy of Sciences of the United

[117] Takahara T, Hara K, Yonezawa K, Sorimachi H, Maeda T. (2006) Nutrient-dependent multimerization of the mammalian target of rapamycin through the N-terminal HEAT repeat region. The Journal of biological chemistry.281(39):28605-14. Epub 2006/07/28. [118] Park IH, Bachmann R, Shirazi H, Chen J. (2002) Regulation of ribosomal S6 kinase 2 by mammalian target of rapamycin. The Journal of biological chemistry.277(35):31423-9.

[119] Lim HK, Choi YA, Park W, Lee T, Ryu SH, et al. (2003) Phosphatidic acid regulates systemic inflammatory responses by modulating the Akt-mammalian target of

myotubes. The Biochemical journal.376(Pt 3):577-86. Epub 2003/09/12.

physiology, biochemistry and pharmacology.144:1-94. Epub 2002/05/04.

[136] Hawley SA, Pan DA, Mustard KJ, Ross L, Bain J, et al. (2005) Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase. Cell metabolism.2(1):9-19. Epub 2005/08/02.

Protein Phosphorylation as a Key Mechanism of mTORC1/2 Signaling Pathways 45

[151] Averous J, Proud CG. (2006) When translation meets transformation: the mTOR story.

[152] Fingar DC, Blenis J. (2004) Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression.

[153] Gingras AC, Raught B, Sonenberg N. (1999) eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annual review of

[154] Nielsen FC, Ostergaard L, Nielsen J, Christiansen J. (1995) Growth-dependent translation of IGF-II mRNA by a rapamycin-sensitive pathway. Nature.377(6547):358-

[155] Rogers GW, Jr., Komar AA, Merrick WC. (2002) eIF4A: the godfather of the DEAD box helicases. Progress in nucleic acid research and molecular biology.72:307-31. Epub

[156] Joshi B, Cai AL, Keiper BD, Minich WB, Mendez R, et al. (1995) Phosphorylation of eukaryotic protein synthesis initiation factor 4E at Ser-209. The Journal of biological

[157] Gingras AC, Raught B, Gygi SP, Niedzwiecka A, Miron M, et al. (2001) Hierarchical phosphorylation of the translation inhibitor 4E-BP1. Genes & development.15(21):2852-

[158] Wang X, Beugnet A, Murakami M, Yamanaka S, Proud CG. (2005) Distinct signaling events downstream of mTOR cooperate to mediate the effects of amino acids and insulin on initiation factor 4E-binding proteins. Mol Cell Biol.25(7):2558-72. Epub

[159] Ferguson G, Mothe-Satney I, Lawrence JC, Jr. (2003) Ser-64 and Ser-111 in PHAS-I are dispensable for insulin-stimulated dissociation from eIF4E. The Journal of biological

[160] Tomoo K, Matsushita Y, Fujisaki H, Abiko F, Shen X, et al. (2005) Structural basis for mRNA Cap-Binding regulation of eukaryotic initiation factor 4E by 4E-binding protein, studied by spectroscopic, X-ray crystal structural, and molecular dynamics simulation

[161] Herbert TP, Tee AR, Proud CG. (2002) The extracellular signal-regulated kinase pathway regulates the phosphorylation of 4E-BP1 at multiple sites. The Journal of

[162] Wang X, Li W, Parra JL, Beugnet A, Proud CG. (2003) The C terminus of initiation factor 4E-binding protein 1 contains multiple regulatory features that influence its

[163] Beugnet A, Wang X, Proud CG. (2003) Target of rapamycin (TOR)-signaling and RAIP motifs play distinct roles in the mammalian TOR-dependent phosphorylation of initiation factor 4E-binding protein 1. The Journal of biological chemistry.278(42):40717-

[164] Avruch J, Belham C, Weng Q, Hara K, Yonezawa K. (2001) The p70 S6 kinase integrates nutrient and growth signals to control translational capacity. Progress in

methods. Biochimica et biophysica acta.1753(2):191-208. Epub 2005/11/08.

function and phosphorylation. Mol Cell Biol.23(5):1546-57. Epub 2003/02/18.

Oncogene.25(48):6423-35. Epub 2006/10/17.

Oncogene.23(18):3151-71. Epub 2004/04/20.

biochemistry.68:913-63. Epub 2000/06/29.

chemistry.270(24):14597-603. Epub 1995/06/16.

chemistry.278(48):47459-65. Epub 2003/09/26.

biological chemistry.277(13):11591-6. Epub 2002/01/19.

molecular and subcellular biology.26:115-54. Epub 2001/09/29.

62. Epub 1995/09/28.

64. Epub 2001/11/03.

22. Epub 2003/08/13.

2002/09/11.

2005/03/16.

[151] Averous J, Proud CG. (2006) When translation meets transformation: the mTOR story. Oncogene.25(48):6423-35. Epub 2006/10/17.

44 Protein Phosphorylation in Human Health

2003/05/29.

Epub 2006/02/18.

2005/07/01.

2008/10/11.

kinase. Cell metabolism.2(1):9-19. Epub 2005/08/02.

growth. Cell.126(5):955-68. Epub 2006/09/09.

[136] Hawley SA, Pan DA, Mustard KJ, Ross L, Bain J, et al. (2005) Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein

[137] Zhang Y, Gao X, Saucedo LJ, Ru B, Edgar BA, et al. (2003) Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins. Nature cell biology.5(6):578-81. Epub

[138] Inoki K, Ouyang H, Zhu T, Lindvall C, Wang Y, et al. (2006) TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell

[139] Arsham AM, Howell JJ, Simon MC. (2003) A novel hypoxia-inducible factorindependent hypoxic response regulating mammalian target of rapamycin and its

[141] Liu L, Cash TP, Jones RG, Keith B, Thompson CB, et al. (2006) Hypoxia-induced energy stress regulates mRNA translation and cell growth. Molecular cell.21(4):521-31.

[142] Brugarolas J, Lei K, Hurley RL, Manning BD, Reiling JH, et al. (2004) Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor

[144] DeYoung MP, Horak P, Sofer A, Sgroi D, Ellisen LW. (2008) Hypoxia regulates TSC1/2-mTOR signaling and tumor suppression through REDD1-mediated 14-3-3

[145] Corradetti MN, Inoki K, Guan KL. (2005) The stress-inducted proteins RTP801 and RTP801L are negative regulators of the mammalian target of rapamycin pathway. The

[146] Schwarzer R, Tondera D, Arnold W, Giese K, Klippel A, et al. (2005) REDD1 integrates hypoxia-mediated survival signaling downstream of phosphatidylinositol 3-kinase.

[147] Bernardi R, Guernah I, Jin D, Grisendi S, Alimonti A, et al. (2006) PML inhibits HIF-1alpha translation and neoangiogenesis through repression of mTOR.

[148] Li Y, Wang Y, Kim E, Beemiller P, Wang CY, et al. (2007) Bnip3 mediates the hypoxiainduced inhibition on mammalian target of rapamycin by interacting with Rheb. The

[149] Wouters BG, Koritzinsky M. (2008) Hypoxia signalling through mTOR and the unfolded protein response in cancer. Nature reviews Cancer.8(11):851-64. Epub

[150] Laplante M, Sabatini DM. (2009) An emerging role of mTOR in lipid biosynthesis.

suppressor complex. Genes & development.18(23):2893-904. Epub 2004/11/17. [143] Sofer A, Lei K, Johannessen CM, Ellisen LW. (2005) Regulation of mTOR and cell growth in response to energy stress by REDD1. Mol Cell Biol.25(14):5834-45. Epub

targets. The Journal of biological chemistry.278(32):29655-60. Epub 2003/06/05. [140] Schneider A, Younis RH, Gutkind JS. (2008) Hypoxia-induced energy stress inhibits the mTOR pathway by activating an AMPK/REDD1 signaling axis in head and neck

squamous cell carcinoma. Neoplasia.10(11):1295-302. Epub 2008/10/28.

shuttling. Genes & development.22(2):239-51. Epub 2008/01/17.

Journal of biological chemistry.280(11):9769-72. Epub 2005/01/06.

Journal of biological chemistry.282(49):35803-13. Epub 2007/10/12.

Current biology : CB.19(22):R1046-52. Epub 2009/12/02.

Oncogene.24(7):1138-49. Epub 2004/12/14.

Nature.442(7104):779-85. Epub 2006/08/18.

[165] Roux PP, Blenis J. (2004) ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiology and molecular biology reviews : MMBR.68(2):320-44. Epub 2004/06/10.

Protein Phosphorylation as a Key Mechanism of mTORC1/2 Signaling Pathways 47

[179] Peterson TR, Sabatini DM. (2005) eIF3: a connecTOR of S6K1 to the translation

[180] Shahbazian D, Roux PP, Mieulet V, Cohen MS, Raught B, et al. (2006) The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity.

[181] Harris TE, Chi A, Shabanowitz J, Hunt DF, Rhoads RE, et al. (2006) mTOR-dependent stimulation of the association of eIF4G and eIF3 by insulin. The EMBO

[182] Alessi DR, Kozlowski MT, Weng QP, Morrice N, Avruch J. (1998) 3-Phosphoinositidedependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in

[183] Pullen N, Dennis PB, Andjelkovic M, Dufner A, Kozma SC, et al. (1998) Phosphorylation and activation of p70s6k by PDK1. Science.279(5351):707-10. Epub

[184] Schalm SS, Blenis J. (2002) Identification of a conserved motif required for mTOR

[185] Ali SM, Sabatini DM. (2005) Structure of S6 kinase 1 determines whether raptor-mTOR or rictor-mTOR phosphorylates its hydrophobic motif site. The Journal of biological

[186] Saitoh M, Pullen N, Brennan P, Cantrell D, Dennis PB, et al. (2002) Regulation of an activated S6 kinase 1 variant reveals a novel mammalian target of rapamycin phosphorylation site. The Journal of biological chemistry.277(22):20104-12. Epub

[187] Isotani S, Hara K, Tokunaga C, Inoue H, Avruch J, et al. (1999) Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase alpha in

[188] Browne GJ, Proud CG. (2002) Regulation of peptide-chain elongation in mammalian cells. European journal of biochemistry / FEBS.269(22):5360-8. Epub 2002/11/09. [189] Carlberg U, Nilsson A, Nygard O. (1990) Functional properties of phosphorylated elongation factor 2. European journal of biochemistry / FEBS.191(3):639-45. Epub

[190] Wang X, Li W, Williams M, Terada N, Alessi DR, et al. (2001) Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase. The EMBO journal.20(16):4370-9. Epub

[191] Ryazanov AG, Ward MD, Mendola CE, Pavur KS, Dorovkov MV, et al. (1997) Identification of a new class of protein kinases represented by eukaryotic elongation factor-2 kinase. Proceedings of the National Academy of Sciences of the United States of

[192] Diggle TA, Seehra CK, Hase S, Redpath NT. (1999) Analysis of the domain structure of elongation factor-2 kinase by mutagenesis. FEBS letters.457(2):189-92. Epub 1999/09/03. [193] Browne GJ, Proud CG. (2004) A Novel mTOR-Regulated Phosphorylation Site in Elongation Factor 2 Kinase Modulates the Activity of the Kinase and Its Binding to

vitro. The Journal of biological chemistry.274(48):34493-8. Epub 1999/11/24.

preinitiation complex. Molecular cell.20(5):655-7. Epub 2005/12/13.

vivo and in vitro. Current biology : CB.8(2):69-81. Epub 1998/03/21.

signaling. Current biology : CB.12(8):632-9. Epub 2002/04/23.

The EMBO journal.25(12):2781-91. Epub 2006/06/10.

journal.25(8):1659-68. Epub 2006/03/17.

chemistry.280(20):19445-8. Epub 2005/04/06.

America.94(10):4884-9. Epub 1997/05/13.

Calmodulin. Molecular and Cellular Biology.24(7):2986-97.

1998/02/21.

2002/03/27.

1990/08/17.

2001/08/14.

[179] Peterson TR, Sabatini DM. (2005) eIF3: a connecTOR of S6K1 to the translation preinitiation complex. Molecular cell.20(5):655-7. Epub 2005/12/13.

46 Protein Phosphorylation in Human Health

reviews : MMBR.68(2):320-44. Epub 2004/06/10.

biology.4(3):251-5. Epub 2002/02/28.

chemistry.280(28):26089-93. Epub 2005/05/21.

kinase. The EMBO journal.17(22):6649-59. Epub 1998/11/21.

kinases. The EMBO journal.23(8):1761-9. Epub 2004/04/09.

pathway. Mol Cell Biol.24(8):3112-24. Epub 2004/04/03.

& development.19(18):2199-211. Epub 2005/09/17.

Biol.24(9):3894-906. Epub 2004/04/15.

[165] Roux PP, Blenis J. (2004) ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiology and molecular biology

[166] Reinhard C, Thomas G, Kozma SC. (1992) A single gene encodes two isoforms of the p70 S6 kinase: activation upon mitogenic stimulation. Proceedings of the National Academy of Sciences of the United States of America.89(9):4052-6. Epub 1992/05/01. [167] Shima H, Pende M, Chen Y, Fumagalli S, Thomas G, et al. (1998) Disruption of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and a new functional S6

[168] Price DJ, Grove JR, Calvo V, Avruch J, Bierer BE. (1992) Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase. Science.257(5072):973-7. Epub 1992/08/14. [169] Montagne J, Stewart MJ, Stocker H, Hafen E, Kozma SC, et al. (1999) Drosophila S6

[170] Radimerski T, Montagne J, Rintelen F, Stocker H, van der Kaay J, et al. (2002) dS6Kregulated cell growth is dPKB/dPI(3)K-independent, but requires dPDK1. Nature cell

[171] Raught B, Peiretti F, Gingras AC, Livingstone M, Shahbazian D, et al. (2004) Phosphorylation of eucaryotic translation initiation factor 4B Ser422 is modulated by S6

[172] Holz MK, Blenis J. (2005) Identification of S6 kinase 1 as a novel mammalian target of rapamycin (mTOR)-phosphorylating kinase. The Journal of biological

[173] Pende M, Um SH, Mieulet V, Sticker M, Goss VL, et al. (2004) S6K1(-/-)/S6K2(-/-) mice exhibit perinatal lethality and rapamycin-sensitive 5'-terminal oligopyrimidine mRNA translation and reveal a mitogen-activated protein kinase-dependent S6 kinase

[174] Tang H, Hornstein E, Stolovich M, Levy G, Livingstone M, et al. (2001) Amino acidinduced translation of TOP mRNAs is fully dependent on phosphatidylinositol 3 kinase-mediated signaling, is partially inhibited by rapamycin, and is independent of

[176] Ruvinsky I, Sharon N, Lerer T, Cohen H, Stolovich-Rain M, et al. (2005) Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis. Genes

[177] Yang HS, Jansen AP, Komar AA, Zheng X, Merrick WC, et al. (2003) The transformation suppressor Pdcd4 is a novel eukaryotic translation initiation factor 4A binding protein that inhibits translation. Mol Cell Biol.23(1):26-37. Epub 2002/12/17. [178] Yang HS, Cho MH, Zakowicz H, Hegamyer G, Sonenberg N, et al. (2004) A novel function of the MA-3 domains in transformation and translation suppressor Pdcd4 is essential for its binding to eukaryotic translation initiation factor 4A. Mol Cell

S6K1 and rpS6 phosphorylation. Mol Cell Biol.21(24):8671-83. Epub 2001/11/20. [175] Stolovich M, Tang H, Hornstein E, Levy G, Cohen R, et al. (2002) Transduction of growth or mitogenic signals into translational activation of TOP mRNAs is fully reliant on the phosphatidylinositol 3-kinase-mediated pathway but requires neither S6K1 nor

rpS6 phosphorylation. Mol Cell Biol.22(23):8101-13. Epub 2002/11/06.

kinase: a regulator of cell size. Science.285(5436):2126-9. Epub 1999/09/25.

[194] Knebel A, Morrice N, Cohen P. (2001) A novel method to identify protein kinase substrates: eEF2 kinase is phosphorylated and inhibited by SAPK4/p38delta. The EMBO journal.20(16):4360-9. Epub 2001/08/14.

Protein Phosphorylation as a Key Mechanism of mTORC1/2 Signaling Pathways 49

[210] Dunlop EA, Hunt DK, Acosta-Jaquez HA, Fingar DC, Tee AR. (2011) ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate

[211] Efeyan A, Sabatini DM. (2010) mTOR and cancer: many loops in one pathway. Current

[212] Egan DF, Shackelford DB, Mihaylova MM, Gelino S, Kohnz RA, et al. (2011) Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy

[213] Lee SB, Kim S, Lee J, Park J, Lee G, et al. (2007) ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase. EMBO reports.8(4):360-5. Epub

[214] Lee S, Comer FI, Sasaki A, McLeod IX, Duong Y, et al. (2005) TOR complex 2 integrates cell movement during chemotaxis and signal relay in Dictyostelium. Molecular biology

[215] Feng J, Park J, Cron P, Hess D, Hemmings BA. (2004) Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase. The Journal of

[216] Guertin DA, Sabatini DM. (2007) Defining the role of mTOR in cancer. Cancer

[217] Pearce LR, Komander D, Alessi DR. (2010) The nuts and bolts of AGC protein kinases.

[218] Schaller MD. (2001) Paxillin: a focal adhesion-associated adaptor protein.

[219] Etienne-Manneville S, Hall A. (2002) Rho GTPases in cell biology.

[220] Oh WJ, Wu CC, Kim SJ, Facchinetti V, Julien LA, et al. (2010) mTORC2 can associate with ribosomes to promote cotranslational phosphorylation and stability of nascent Akt

[221] Roberts PJ, Der CJ. (2007) Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene.26(22):3291-310. Epub 2007/05/15. [222] Murphy LO, Blenis J. (2006) MAPK signal specificity: the right place at the right time.

[223] Dalby KN, Morrice N, Caudwell FB, Avruch J, Cohen P. (1998) Identification of regulatory phosphorylation sites in mitogen-activated protein kinase (MAPK)-activated protein kinase-1a/p90rsk that are inducible by MAPK. The Journal of biological

[224] Anjum R, Blenis J. (2008) The RSK family of kinases: emerging roles in cellular signalling. Nature reviews Molecular cell biology.9(10):747-58. Epub 2008/09/25. [225] Roux PP, Ballif BA, Anjum R, Gygi SP, Blenis J. (2004) Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 ribosomal S6 kinase. Proceedings of the National Academy of Sciences of the

[226] Ma L, Chen Z, Erdjument-Bromage H, Tempst P, Pandolfi PP. (2005) Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and

binding. Autophagy.7(7):737-47. Epub 2011/04/05.

of the cell.16(10):4572-83. Epub 2005/08/05.

Oncogene.20(44):6459-72. Epub 2001/10/19.

Nature.420(6916):629-35. Epub 2002/12/13.

chemistry.273(3):1496-505. Epub 1998/01/27.

Cell.12(1):9-22. Epub 2007/07/07.

biological chemistry.279(39):41189-96. Epub 2004/07/21.

Nature reviews Molecular cell biology.11(1):9-22. Epub 2009/12/23.

polypeptide. The EMBO journal.29(23):3939-51. Epub 2010/11/04.

Trends in biochemical sciences.31(5):268-75. Epub 2006/04/11.

United States of America.101(37):13489-94. Epub 2004/09/03.

cancer pathogenesis. Cell.121(2):179-93. Epub 2005/04/27.

2007/03/10.

opinion in cell biology.22(2):169-76. Epub 2009/12/01.

sensing to mitophagy. Science.331(6016):456-61. Epub 2011/01/06.

[210] Dunlop EA, Hunt DK, Acosta-Jaquez HA, Fingar DC, Tee AR. (2011) ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding. Autophagy.7(7):737-47. Epub 2011/04/05.

48 Protein Phosphorylation in Human Health

2009/02/20.

Epub 2009/12/10.

2011/01/25.

2009/07/15.

2010/07/20.

journal.20(16):4360-9. Epub 2001/08/14.

the cell.20(7):1992-2003. Epub 2009/02/20.

cell.43(4):572-85. Epub 2011/08/23.

[194] Knebel A, Morrice N, Cohen P. (2001) A novel method to identify protein kinase substrates: eEF2 kinase is phosphorylated and inhibited by SAPK4/p38delta. The EMBO

[195] Tsang CK, Liu H, Zheng XF. (2010) mTOR binds to the promoters of RNA polymerase

[196] Dobashi Y, Watanabe Y, Miwa C, Suzuki S, Koyama S. (2011) Mammalian target of rapamycin: a central node of complex signaling cascades. International journal of

[197] Klionsky DJ. (2007) Autophagy: from phenomenology to molecular understanding in less than a decade. Nature reviews Molecular cell biology.8(11):931-7. Epub 2007/08/23. [198] Levine B, Klionsky DJ. (2004) Development by self-digestion: molecular mechanisms and biological functions of autophagy. Developmental cell.6(4):463-77. Epub 2004/04/08. [199] Liu JJ, Lin M, Yu JY, Liu B, Bao JK. (2011) Targeting apoptotic and autophagic pathways for cancer therapeutics. Cancer letters.300(2):105-14. Epub 2010/11/03. [200] Chang YY, Neufeld TP. (2009) An Atg1/Atg13 complex with multiple roles in TORmediated autophagy regulation. Molecular biology of the cell.20(7):2004-14. Epub

[201] Jung CH, Jun CB, Ro SH, Kim YM, Otto NM, et al. (2009) ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery. Molecular biology of