demonstrate that, despite high similarity in the recognition sequences of basophilic serine/threonine protein kinases, specific kinase signaling can modulate membrane protein trafficking through 14-3-3 binding. Further search of the human protein database for this Cterminal 14-3-3-binding motif has identified several candidate membrane proteins that would interact with 14-3-3. These include GPR15, an orphan GPCR that serves for a coreceptor for human immunodeficiency virus (HIV) entry (Farzan *et al.*, 1997). GPR15, which has a C-terminal sequence of RRRKRSVSL-COOH, was indeed confirmed to bind 14-3-3 and this binding absolutely required phosphorylation of penultimate serine359. A recent study by Okamoto and Shikano reported that alanine mutation of the serine359 resulted in substantial ER localization of GPR15 and this was mediated by the upstream arginine residues at amino acids 352 and 354, which constitute a COPI-binding RXR motif (Okamoto & Shikano, 2011). These results suggested that mode III binding of 14-3-3 to the receptor C-terminus physically occludes the adjacent RXR motif from the access by COPI, similar to the cases for TASK channels. Thus, a non-biased screening has led to the identification of 14-3-3 function as a key switch that converts phosphorylation signal to the sorting of membrane proteins by modulating the activity of an ER localization signal.

The masking effect of 14-3-3 on the cell surface transport is not restricted to the mode III Cterminal binding. The cytoplasmic tail of ADAM22, a member of ADAM (a disintegrin and metalloprotease domain) protein family, contains two internal 14-3-3 binding sites and three RXR-type ER-localization signals that overlap with both of the 14-3-3 protein binding sites. Mutations in both 14-3-3 binding sites inhibited surface expression of ADAM22, while deletion of both RXR motif and 14-3-3 binding sites restored the surface expression (Godde *et al.*, 2006). Although this study did not investigate COPI interaction with ADAM22, the results suggest the possibility that 14-3-3 binding inhibited the COPI-dependent ER localization activity of RXR signal.

The cell surface transport of a gap junction protein connexin 43 is also promoted by 14-3-3 binding to its internal mode I binding site (RASSRP) (Park *et al.*, 2007). The authors have found that Akt phosphorylates the serine373 in this site in the epidermal growth factor (EGF) stimulated cell. Although not characterized yet, the existence of an overlapping RXR motif (RPR) suggests a possible masking effect of 14-3-3 on the cell surface transport of connexin 43.

NMDA receptors are tetramers composed of homologous subunits (NR1; NR2A-D; NR3A-B (Cull-Candy & Leszkiewicz, 2004). There are multiple NR2 subunits, each with unique spatio-temporal expression patterns, ensuring functional diversity of NMDA receptors. Recently, cerebellar NR2C subunit was found to be directly phosphorylated by Akt at the sequence that conforms to mode I 14-3-3 binding motif (RPRHASLP) (Chen & Roche, 2009). The Akt phosphorylation induced by insulin growth factor (IGF-1) results in the recruitment of 14-3-3 and the increase of cell surface expression of NMDA receptors. Although the causative role of 14-3-3 in surface transport remains unclear in this study, the presence of ER localization signals (Horak & Wenthold, 2009) in the NR1 subunit and the obligatory assembly of NR2 with NR1 for functional NMDA receptor suggest a possible mechanism where Akt-induced 14-3-3 binding to NR2C attenuates the activity of ER localization signals in NR1 by physically occluding them.

Interestingly, 14-3-3s have also been reported to bind directly to the RXR motif itself in a phosphorylation-independent manner. By using an artificial multimer of the distal C-

Phosphorylation-Regulated Cell Surface Expression of Membrane Proteins 175

proteins may provide for a mechanism by which nAChRs containing only specific subunits are recruited to postsynaptic clusters and may stabilise them there. In both of the above studies, it is still not clear whether and how a 14-3-3 dimer binds to two separate targets with each monomer. Multiple proteomic screenings have revealed that 14-3-3s form complex with a large number of proteins closely involved in vesicular trafficking such as motor proteins, coat proteins, and GTPase regulators (Mrowiec & Schwappach, 2006). Therefore it is conceivable that 14-3-3 proteins do modulate membrane trafficking by serving as a scaffold that connects cargo proteins with cellular transport machineries.

Then, does a 14-3-3 dimer really bind to two different proteins at the same time? It seems somewhat unlikely that there would be frequent occasions where a 14-3-3 dimer binds two separate targets via each canonical ligand-binding groove, unless those target proteins happen to be already close enough to each other and in such position that both of their phosphorylated binding sites can be accommodated by the binding grooves of the same 14- 3-3 dimer, whose core is a rigid and unyielding structure (Obsil *et al.*, 2001), but not by the neighboring 14-3-3 dimers. Crystal structure of 14-3-3*ζ* : AANAT revealed that in addition to the phosphorylation-dependent interaction through its canonical ligand-binding groove, 14- 3-3 also makes extensive contacts with AANAT via other regions of the 14-3-3 channel, although these contacts must be insufficient to form a stable complex (Obsil *et al.*, 2001). Moreover, a recent finding by Barry *et al*. demonstrated that 14-3-3 can directly interact with other proteins outside of the canonical binding groove, providing a possible molecular basis for the scaffolding function of 14-3-3 (Barry *et al.*, 2009). 14-3-3 undergoes phosphorylation at tyrosine179 upon cytokine stimulation and this leads to the binding of Shc protein through its SH2 domain that recognizes phosphorylated tyrosine. This 14-3-3/Shc complex is required for the recruitment of a phosphatidylinositol 3-kinase (PI3K) signaling complex and the regulation of cell survival in response to cytokine. Although this study did not describe whether Shc-bound 14-3-3 proteins bind to any serine/threonine-phosphorylated targets, the result suggests that 14-3-3/Shc scaffolds can act as multivalent signaling nodes for the integration of both phosphoserine/threonine and phosphotyrosine pathways to regulate specific cellular responses. Thus, interaction of 14-3-3 with proteins via noncanonical binding sites of 14-3-3 should contribute to the diversity of its roles in a wide

In addition, the propensity of the different 14-3-3 isoforms to form homo- or hetero-dimers may confer additional specificity to the scaffolding roles. Those regions of the 14-3-3 protein which vary between the isoforms are primarily located on the surface of the protein. Therefore, the specificity of interaction of 14-3-3 isoforms with diverse target proteins may involve the outer surface of the protein. For instance, C-termini of 14-3-3 proteins are most divergent and hence most likely to contain isoform-specific structural determinants (Williams *et al*., 2011). Identification of more protein complexes whose assembly requires 14- 3-3 is necessary to gain more mechanistic insights into the scaffolding function of 14-3-3.

Another mechanism by which 14-3-3 proteins are thought to exert their effect on their targets are conformational 'clamping'. Clamping can occur when a 14-3-3 dimer binds two sites on the same target protein. A synthetic phosphopeptide with two tandem 14-3-3 consensus motifs binds over 30-fold more tightly than the same peptide containing only a

variety of biological pathways including membrane trafficking.

**2.3.3 Clamping**

terminus of Kir6.2 channel in pull-down assays from cytosolic cellular extracts, Yuan *et al.* showed that 14-3-3 proteins preferentially bound to the RXR motif (RSRR) in the oligomerized form (Yuan *et al.*, 2003). This interaction was sufficient to allow the exit of a multimeric reporter protein carrying this motif from the ER and promote the subsequent transport to the cell surface. Although the precise role of the 14-3-3 binding in the cell surface transport of octameric KATP channel consisting of four Kir6.2 and four SUR1 seems to require further investigation (Heusser *et al.*, 2006), this study demonstrated the possibility that 14-3-3 serves for a constitutive check point of ER protein quality control which ensures the cell surface delivery of functional multimeric membrane proteins by preferentially inactivating the ER localisation signals on the properly oligomerized subunits.

#### **2.3.2 Scaffolding**

The stable dimeric structure immediately suggests that 14-3-3s might serve as a simple 'scaffold', where two different target proteins bind simultaneously to each monomer of the same 14-3-3 dimer. Indeed, 14-3-3 proteins are often referred to as 'scaffolding proteins' in the literature. However, while 14-3-3s are components of multiprotein complexes (Munday *et al.*, 2000; Pnueli *et al.*, 2001; Widen *et al.*, 2000), the evidence showing the 14-3-3 dimers acting as an intermolecular bridge between two different substrates had been limited to several earlier studies including those reporting the pairings involving Raf-1, namely Raf-1 and Bcr (B-cell receptor) (Braselmann & McCormick, 1995), Raf-1 and A20 (Vincenz & Dixit, 1996) and Raf-1 and PKC*ζ* (Van Der Hoeven *et al.*, 2000). Nevertheless, in the context of membrane protein trafficking, some recent studies implicate the scaffolding role of 14-3-3 in promoting the cell surface expression of membrane proteins.

14-3-3 promotes the ER export of N-cadherin through coupling the N-cadherin/-catenin/PX-RICS complex to the microtubule-based motor proteins dynein/dynactin (Nakamura *et al.*, 2010). 14-3-3ζ or 14-3-3θ directly interacts in a phosphorylation-dependent manner with a mode I site (RSKSDP) of PX-RICS, a β-catenin-interacting GTPase-activating protein for Cdc42, and this seems to facilitate ER to Golgi trafficking by association of N-cadherin/βcatenin cargo with minus-end motor proteins dynein/dynactin. This results in the increased localization of N-cadherin/β-catenin at cell-cell contact sites. The authors have also shown that CaMKII is responsible for direct phosphorylation of PX-RICS and the subsequent 14-3-3 binding by using *in vitro* phosphorylation assay and siRNA knockdown of CaMKII (Nakamura *et al.*, 2010). It is of note that a similar scaffolding function has been reported for a PDZ protein that couples a cargo receptor and a motor protein which mediates microtubule-based trafficking. PDZ domain of mLin-10 directly interacts with the Cterminal PDZ-binding motif of a neuron-specific plus-end molecular motor KIF-17. mLin-10 also forms a complex with its family members mLin-2 and mLin-7, which in turn interact with PDZ-binding motif of NMDA receptor subunit 2B (Setou *et al.*, 2000).

The interactions between the α3 subunit of the nicotinic acetylcholine receptor (nAChR) and a multi-subunit cytoskeletal-anchoring complex provide another evidence suggesting possible scaffolding role of 14-3-3 (Rosenberg *et al.*, 2008). APC (adenomatous polyposis coli) organizes a multi-protein postsynaptic complex that targets α3nAChRs to synapses. APC interaction with the microtubule plus-end binding protein EB1 is essential for α3nAChR surface membrane insertion and stabilization. 14-3-3 directly interacts with α3 subunit in a phosphorylation-dependent manner and also forms complex with APC. Thus, 14-3-3 proteins may provide for a mechanism by which nAChRs containing only specific subunits are recruited to postsynaptic clusters and may stabilise them there. In both of the above studies, it is still not clear whether and how a 14-3-3 dimer binds to two separate targets with each monomer. Multiple proteomic screenings have revealed that 14-3-3s form complex with a large number of proteins closely involved in vesicular trafficking such as motor proteins, coat proteins, and GTPase regulators (Mrowiec & Schwappach, 2006). Therefore it is conceivable that 14-3-3 proteins do modulate membrane trafficking by serving as a scaffold that connects cargo proteins with cellular transport machineries.

Then, does a 14-3-3 dimer really bind to two different proteins at the same time? It seems somewhat unlikely that there would be frequent occasions where a 14-3-3 dimer binds two separate targets via each canonical ligand-binding groove, unless those target proteins happen to be already close enough to each other and in such position that both of their phosphorylated binding sites can be accommodated by the binding grooves of the same 14- 3-3 dimer, whose core is a rigid and unyielding structure (Obsil *et al.*, 2001), but not by the neighboring 14-3-3 dimers. Crystal structure of 14-3-3*ζ* : AANAT revealed that in addition to the phosphorylation-dependent interaction through its canonical ligand-binding groove, 14- 3-3 also makes extensive contacts with AANAT via other regions of the 14-3-3 channel, although these contacts must be insufficient to form a stable complex (Obsil *et al.*, 2001). Moreover, a recent finding by Barry *et al*. demonstrated that 14-3-3 can directly interact with other proteins outside of the canonical binding groove, providing a possible molecular basis for the scaffolding function of 14-3-3 (Barry *et al.*, 2009). 14-3-3 undergoes phosphorylation at tyrosine179 upon cytokine stimulation and this leads to the binding of Shc protein through its SH2 domain that recognizes phosphorylated tyrosine. This 14-3-3/Shc complex is required for the recruitment of a phosphatidylinositol 3-kinase (PI3K) signaling complex and the regulation of cell survival in response to cytokine. Although this study did not describe whether Shc-bound 14-3-3 proteins bind to any serine/threonine-phosphorylated targets, the result suggests that 14-3-3/Shc scaffolds can act as multivalent signaling nodes for the integration of both phosphoserine/threonine and phosphotyrosine pathways to regulate specific cellular responses. Thus, interaction of 14-3-3 with proteins via noncanonical binding sites of 14-3-3 should contribute to the diversity of its roles in a wide variety of biological pathways including membrane trafficking.

In addition, the propensity of the different 14-3-3 isoforms to form homo- or hetero-dimers may confer additional specificity to the scaffolding roles. Those regions of the 14-3-3 protein which vary between the isoforms are primarily located on the surface of the protein. Therefore, the specificity of interaction of 14-3-3 isoforms with diverse target proteins may involve the outer surface of the protein. For instance, C-termini of 14-3-3 proteins are most divergent and hence most likely to contain isoform-specific structural determinants (Williams *et al*., 2011). Identification of more protein complexes whose assembly requires 14- 3-3 is necessary to gain more mechanistic insights into the scaffolding function of 14-3-3.

#### **2.3.3 Clamping**

174 Protein Kinases

terminus of Kir6.2 channel in pull-down assays from cytosolic cellular extracts, Yuan *et al.* showed that 14-3-3 proteins preferentially bound to the RXR motif (RSRR) in the oligomerized form (Yuan *et al.*, 2003). This interaction was sufficient to allow the exit of a multimeric reporter protein carrying this motif from the ER and promote the subsequent transport to the cell surface. Although the precise role of the 14-3-3 binding in the cell surface transport of octameric KATP channel consisting of four Kir6.2 and four SUR1 seems to require further investigation (Heusser *et al.*, 2006), this study demonstrated the possibility that 14-3-3 serves for a constitutive check point of ER protein quality control which ensures the cell surface delivery of functional multimeric membrane proteins by preferentially

The stable dimeric structure immediately suggests that 14-3-3s might serve as a simple 'scaffold', where two different target proteins bind simultaneously to each monomer of the same 14-3-3 dimer. Indeed, 14-3-3 proteins are often referred to as 'scaffolding proteins' in the literature. However, while 14-3-3s are components of multiprotein complexes (Munday *et al.*, 2000; Pnueli *et al.*, 2001; Widen *et al.*, 2000), the evidence showing the 14-3-3 dimers acting as an intermolecular bridge between two different substrates had been limited to several earlier studies including those reporting the pairings involving Raf-1, namely Raf-1 and Bcr (B-cell receptor) (Braselmann & McCormick, 1995), Raf-1 and A20 (Vincenz & Dixit, 1996) and Raf-1 and PKC*ζ* (Van Der Hoeven *et al.*, 2000). Nevertheless, in the context of membrane protein trafficking, some recent studies implicate the scaffolding role of 14-3-3 in

14-3-3 promotes the ER export of N-cadherin through coupling the N-cadherin/-catenin/PX-RICS complex to the microtubule-based motor proteins dynein/dynactin (Nakamura *et al.*, 2010). 14-3-3ζ or 14-3-3θ directly interacts in a phosphorylation-dependent manner with a mode I site (RSKSDP) of PX-RICS, a β-catenin-interacting GTPase-activating protein for Cdc42, and this seems to facilitate ER to Golgi trafficking by association of N-cadherin/βcatenin cargo with minus-end motor proteins dynein/dynactin. This results in the increased localization of N-cadherin/β-catenin at cell-cell contact sites. The authors have also shown that CaMKII is responsible for direct phosphorylation of PX-RICS and the subsequent 14-3-3 binding by using *in vitro* phosphorylation assay and siRNA knockdown of CaMKII (Nakamura *et al.*, 2010). It is of note that a similar scaffolding function has been reported for a PDZ protein that couples a cargo receptor and a motor protein which mediates microtubule-based trafficking. PDZ domain of mLin-10 directly interacts with the Cterminal PDZ-binding motif of a neuron-specific plus-end molecular motor KIF-17. mLin-10 also forms a complex with its family members mLin-2 and mLin-7, which in turn interact

The interactions between the α3 subunit of the nicotinic acetylcholine receptor (nAChR) and a multi-subunit cytoskeletal-anchoring complex provide another evidence suggesting possible scaffolding role of 14-3-3 (Rosenberg *et al.*, 2008). APC (adenomatous polyposis coli) organizes a multi-protein postsynaptic complex that targets α3nAChRs to synapses. APC interaction with the microtubule plus-end binding protein EB1 is essential for α3nAChR surface membrane insertion and stabilization. 14-3-3 directly interacts with α3 subunit in a phosphorylation-dependent manner and also forms complex with APC. Thus, 14-3-3

inactivating the ER localisation signals on the properly oligomerized subunits.

promoting the cell surface expression of membrane proteins.

with PDZ-binding motif of NMDA receptor subunit 2B (Setou *et al.*, 2000).

**2.3.2 Scaffolding**

Another mechanism by which 14-3-3 proteins are thought to exert their effect on their targets are conformational 'clamping'. Clamping can occur when a 14-3-3 dimer binds two sites on the same target protein. A synthetic phosphopeptide with two tandem 14-3-3 consensus motifs binds over 30-fold more tightly than the same peptide containing only a

Phosphorylation-Regulated Cell Surface Expression of Membrane Proteins 177

coiled-coil dimerization, triggers cell surface trafficking of GABAB receptors. Thus, it is interesting to speculate that clamping of two different 14-3-3 binding sites, either within the same target protein or in two neighboring proteins, might lead to the placement of RXR motifs in a non-functional zone such as membrane proximity and suppress ER localization

**2.3.4 Other mechanisms by which 14-3-3s modulate membrane protein trafficking**

Modulation of membrane protein localization by 14-3-3s is not necessarily mediated by their binding to the cargo protein itself. It is not surprising that 14-3-3 binding to any cellular machinery proteins involved in biosynthetic pathways would affect their functions and

The best characterized mechanism of this type involves the interaction of 14-3-3 with AS160, a Rab GTPase-activating protein (Rab-GAP). AS160 is an Akt substrate whose phosphorylation contributes to the recruitment of GLUT4 transporters to adipocyte plasma membrane in response to insulin (Watson & Pessin, 2006). It maintains Rab proteins with which it associates in their inactive, GDP-bound states. Several evidences implicate Akt-mediated AS160 phosphorylation and the subsequent 14-3-3 binding in the recruitment of the GLUT4 glucose transporter to the cell surface of adipocytes (Ramm *et al.*, 2006; Watson & Pessin, 2006). Insulin stimulation leads to the recruitment of Akt to the plasma membrane where it gets activated and then phosphorylate AS160 at serine341 and threonine642. It is thought that the binding of 14-3-3 to AS160 inhibits its Rab-GAP activity toward substrate Rabs (Ishikura *et al.*, 2007), which stabilizes them at GTP-bound active form and thereby facilitate the trafficking of cargo vesicles. Interestingly, insulin-dependent cell surface transport of GLUT4 is known to be primarily mediated by Akt isoform 2 (Akt2) but not Akt1 in adipocytes (Bae *et al.*, 2003; Cho *et al.*, 2001). A recent study by Gonzalez and McGraw demonstrated that upon insulin stimulation, Akt2 is able to remain associated with plasma membrane longer than Akt1 does and this leads to the Akt2-specific phosphorylation of AS160, which is necessary for GLUT4 trafficking (Gonzalez & McGraw, 2009). It will be interesting to investigate whether and how this Akt isoform-specific phosphorylation of AS160 regulates 14-3-3

The surface expression of the epithelial sodium channel (ENaC) seems to be regulated in a similar manner that involves AS160-14-3-3 interaction (Liang *et al.*, 2010). Aldosterone stimulation of renal epithelial cell induces the expression of serum- and glucocorticoidinduced kinase (SGK1) that increases the cell surface expression of ENaC and Na absorption (Bhalla *et al.*, 2006). Aldosterone also induces the expression of two 14-3-3 protein isoforms, β and ε (Liang *et al.*, 2006). Liang *et al*. reported that SGK1, which is the downstream kinase of PI3K and shares with Akt the recognition motif of RXRXXS/T (Tessier & Woodgett, 2006), phosphorylates AS160 upon aldosterone stimulation and this recruits the induced 14-3-3 isoforms (Liang *et al.*, 2010), similar to the insulin-induced Akt phosphorylation of AS160. Inhibition of ENaC surface transport by expression of AS160 carrying mutations in SGK1 target sites suggests that 14-3-3 downregulates AS160 function to eventually promote surface transport of ENaC and augment Na absorption in response

of target membrane proteins.

binding.

to aldosterone.

thereby affect the sorting of the cargo protein.

single motif (Yaffe *et al.*, 1997). A number of 14-3-3-binding proteins, including Raf-1 (Muslin *et al.*, 1996), AANAT (Ganguly *et al.*, 2005), ADAM22 (Godde *et al.*, 2006), tyrosine hydroxylase (Toska *et al.*, 2002), and Ndel1 (Johnson *et al.*, 2010), contain two phosphorylated sites that are implicated in 14-3-3 binding, and are separated by polypeptides of various lengths. It has been postulated that one site called the 'gatekeeper' is indispensable for a stable 14-3-3 interaction, whereas a second site 'enhances' the interaction, but has too weak an affinity to bind 14-3-3 alone (Yaffe, 2002). In the case of AANAT, the gatekeeper residue is phosphorylated threonine31. Binding of the gatekeeper leads to binding of a second low-affinity site, in this case phosphorylated serine205, which reflects both the intrinsic affinity of that site and the 'high local concentration induced by its proximity' (Ganguly *et al.*, 2005; Yaffe, 2002). This dual-site binding of AANAT to 14-3-3 provides optimal conformation of the enzyme for high-affinity binding of the substrate arylalkylamine.

14-3-3 clamping can also occur when a 14-3-3 dimer binds two neighboring target proteins. 14-3-3 proteins activate the plant plasma membrane H(+)-ATPase (PMA2) by binding to its C-terminal autoinhibitory domain. This interaction requires phosphorylation of a C-terminal mode III recognition motif as well as an adjacent span of 52 amino acids. X-ray diffraction studies using crystals of 14-3-3 in complex with the entire binding motif of the PMA2 have shown that each 14-3-3 dimer simultaneously binds to two H+-ATPase molecules. The 3D reconstruction of the purified H(+)-ATPase/14-3-3 complex demonstrated a hexagonal structure consisting of six PMA2 subunits and six 14-3-3 proteins (Ottmann *et al.*, 2007). Thus, a rigid 14-3-3 'clamps' stabilize the dodecameric compex in the active conformation where C-terminal auto-inhibitory domain of PMA would be displaced.

With regards to membrane protein trafficking, clamping activity of 14-3-3 proteins has been much less well understood. This is largely due to the, as yet, small number of studies in this research area and the lack of high-resolution structure of the 14-3-3/target membrane protein complex, which are also true for 'masking' and 'scaffolding' mechanisms. However, the ability of 14-3-3 to change conformation of target proteins suggests the possibility that such conformational change will lead to a new interaction of the target with proteins that are involved in protein trafficking. This way 14-3-3 might indirectly exert the scaffolding function that eventually modulates the protein sorting of the client protein. Alternatively, instead of direct 'masking' of RXR motif by 14-3-3, 14-3-3 binding might force the target protein into the conformation where the RXR motif will be occluded. The RXR motif was previously found to have its functional 'zoning' in relation to the transmembrane region in the context of a reporter CD4 protein (Shikano & Li, 2003). This notion was based on the observation that the ER localization activity of RXR motif was lost when it was positioned proximal to the transmembrane region of CD4. This zoning model was supported by the later study on the gamma-aminobutyric acid type B (GABAB) receptor (Gassmann *et al.*, 2005). An RXR motif (RSRR) is responsible for the ER retrieval of the GABAB1 subunits that were not properly assembled with GABAB2 subunits. It had been thought that coiled-coil interaction of the GABAB1 with GABAB2 will shield RSRR signal on GABAB1. However, closer positioning of RSRR signal to the membrane region drastically reduced its effectiveness and also functional ectopic RSRR signals in GABAB1 were efficiently inactivated by the GABAB2 subunit in the absence of coiled-coil dimerization (Gassmann *et al.*, 2005). These results were consistent with a model in which removal of RSRR from its functionally active zone, rather than its direct shielding by

single motif (Yaffe *et al.*, 1997). A number of 14-3-3-binding proteins, including Raf-1 (Muslin *et al.*, 1996), AANAT (Ganguly *et al.*, 2005), ADAM22 (Godde *et al.*, 2006), tyrosine hydroxylase (Toska *et al.*, 2002), and Ndel1 (Johnson *et al.*, 2010), contain two phosphorylated sites that are implicated in 14-3-3 binding, and are separated by polypeptides of various lengths. It has been postulated that one site called the 'gatekeeper' is indispensable for a stable 14-3-3 interaction, whereas a second site 'enhances' the interaction, but has too weak an affinity to bind 14-3-3 alone (Yaffe, 2002). In the case of AANAT, the gatekeeper residue is phosphorylated threonine31. Binding of the gatekeeper leads to binding of a second low-affinity site, in this case phosphorylated serine205, which reflects both the intrinsic affinity of that site and the 'high local concentration induced by its proximity' (Ganguly *et al.*, 2005; Yaffe, 2002). This dual-site binding of AANAT to 14-3-3 provides optimal conformation of the enzyme for

14-3-3 clamping can also occur when a 14-3-3 dimer binds two neighboring target proteins. 14-3-3 proteins activate the plant plasma membrane H(+)-ATPase (PMA2) by binding to its C-terminal autoinhibitory domain. This interaction requires phosphorylation of a C-terminal mode III recognition motif as well as an adjacent span of 52 amino acids. X-ray diffraction studies using crystals of 14-3-3 in complex with the entire binding motif of the PMA2 have shown that each 14-3-3 dimer simultaneously binds to two H+-ATPase molecules. The 3D reconstruction of the purified H(+)-ATPase/14-3-3 complex demonstrated a hexagonal structure consisting of six PMA2 subunits and six 14-3-3 proteins (Ottmann *et al.*, 2007). Thus, a rigid 14-3-3 'clamps' stabilize the dodecameric compex in the active conformation

With regards to membrane protein trafficking, clamping activity of 14-3-3 proteins has been much less well understood. This is largely due to the, as yet, small number of studies in this research area and the lack of high-resolution structure of the 14-3-3/target membrane protein complex, which are also true for 'masking' and 'scaffolding' mechanisms. However, the ability of 14-3-3 to change conformation of target proteins suggests the possibility that such conformational change will lead to a new interaction of the target with proteins that are involved in protein trafficking. This way 14-3-3 might indirectly exert the scaffolding function that eventually modulates the protein sorting of the client protein. Alternatively, instead of direct 'masking' of RXR motif by 14-3-3, 14-3-3 binding might force the target protein into the conformation where the RXR motif will be occluded. The RXR motif was previously found to have its functional 'zoning' in relation to the transmembrane region in the context of a reporter CD4 protein (Shikano & Li, 2003). This notion was based on the observation that the ER localization activity of RXR motif was lost when it was positioned proximal to the transmembrane region of CD4. This zoning model was supported by the later study on the gamma-aminobutyric acid type B (GABAB) receptor (Gassmann *et al.*, 2005). An RXR motif (RSRR) is responsible for the ER retrieval of the GABAB1 subunits that were not properly assembled with GABAB2 subunits. It had been thought that coiled-coil interaction of the GABAB1 with GABAB2 will shield RSRR signal on GABAB1. However, closer positioning of RSRR signal to the membrane region drastically reduced its effectiveness and also functional ectopic RSRR signals in GABAB1 were efficiently inactivated by the GABAB2 subunit in the absence of coiled-coil dimerization (Gassmann *et al.*, 2005). These results were consistent with a model in which removal of RSRR from its functionally active zone, rather than its direct shielding by

high-affinity binding of the substrate arylalkylamine.

where C-terminal auto-inhibitory domain of PMA would be displaced.

coiled-coil dimerization, triggers cell surface trafficking of GABAB receptors. Thus, it is interesting to speculate that clamping of two different 14-3-3 binding sites, either within the same target protein or in two neighboring proteins, might lead to the placement of RXR motifs in a non-functional zone such as membrane proximity and suppress ER localization of target membrane proteins.

#### **2.3.4 Other mechanisms by which 14-3-3s modulate membrane protein trafficking**

Modulation of membrane protein localization by 14-3-3s is not necessarily mediated by their binding to the cargo protein itself. It is not surprising that 14-3-3 binding to any cellular machinery proteins involved in biosynthetic pathways would affect their functions and thereby affect the sorting of the cargo protein.

The best characterized mechanism of this type involves the interaction of 14-3-3 with AS160, a Rab GTPase-activating protein (Rab-GAP). AS160 is an Akt substrate whose phosphorylation contributes to the recruitment of GLUT4 transporters to adipocyte plasma membrane in response to insulin (Watson & Pessin, 2006). It maintains Rab proteins with which it associates in their inactive, GDP-bound states. Several evidences implicate Akt-mediated AS160 phosphorylation and the subsequent 14-3-3 binding in the recruitment of the GLUT4 glucose transporter to the cell surface of adipocytes (Ramm *et al.*, 2006; Watson & Pessin, 2006). Insulin stimulation leads to the recruitment of Akt to the plasma membrane where it gets activated and then phosphorylate AS160 at serine341 and threonine642. It is thought that the binding of 14-3-3 to AS160 inhibits its Rab-GAP activity toward substrate Rabs (Ishikura *et al.*, 2007), which stabilizes them at GTP-bound active form and thereby facilitate the trafficking of cargo vesicles. Interestingly, insulin-dependent cell surface transport of GLUT4 is known to be primarily mediated by Akt isoform 2 (Akt2) but not Akt1 in adipocytes (Bae *et al.*, 2003; Cho *et al.*, 2001). A recent study by Gonzalez and McGraw demonstrated that upon insulin stimulation, Akt2 is able to remain associated with plasma membrane longer than Akt1 does and this leads to the Akt2-specific phosphorylation of AS160, which is necessary for GLUT4 trafficking (Gonzalez & McGraw, 2009). It will be interesting to investigate whether and how this Akt isoform-specific phosphorylation of AS160 regulates 14-3-3 binding.

The surface expression of the epithelial sodium channel (ENaC) seems to be regulated in a similar manner that involves AS160-14-3-3 interaction (Liang *et al.*, 2010). Aldosterone stimulation of renal epithelial cell induces the expression of serum- and glucocorticoidinduced kinase (SGK1) that increases the cell surface expression of ENaC and Na absorption (Bhalla *et al.*, 2006). Aldosterone also induces the expression of two 14-3-3 protein isoforms, β and ε (Liang *et al.*, 2006). Liang *et al*. reported that SGK1, which is the downstream kinase of PI3K and shares with Akt the recognition motif of RXRXXS/T (Tessier & Woodgett, 2006), phosphorylates AS160 upon aldosterone stimulation and this recruits the induced 14-3-3 isoforms (Liang *et al.*, 2010), similar to the insulin-induced Akt phosphorylation of AS160. Inhibition of ENaC surface transport by expression of AS160 carrying mutations in SGK1 target sites suggests that 14-3-3 downregulates AS160 function to eventually promote surface transport of ENaC and augment Na absorption in response to aldosterone.

Phosphorylation-Regulated Cell Surface Expression of Membrane Proteins 179

signaling event facilitate the release of 14-3-3s from client proteins? In the context of protein trafficking, 14-3-3 is known to modulate sorting of the client proteins by various different mechanisms. In many of the cases where surface membrane transport is promoted by 14-3-3 binding, 14-3-3 seems to impinge on the early step of ER-to-Golgi trafficking. However, except for a very small number of studies (Godde *et al.*, 2006; Okamoto & Shikano, 2011), it is not understood if 14-3-3 proteins remain bound to the client all along the trip to the cell surface or dissociate in any particular step of the vesicular trafficking by phosphatase activity. These problems make us realize that we still do not know enough about the biology of kinases and phosphatases, especially the spatio-temporal regulation of their activities in

Another important question that has been long discussed but not fully addressed is whether and how 14-3-3 isoforms play specific roles. The complete sequence conservation in the observed ligand-binding regions of 14-3-3 would support the hypothesis that there may be little isoform specificity in the interaction between 14-3-3 and client proteins; therefore isoform-specific function of 14-3-3 may result either from subcellular localization (Paul *et al.*, 2005; van Hemert *et al.*, 2004) or transcriptional regulation (Liang *et al.*, 2006) of particular isotypes rather than from inherent differences in their ability to bind to particular ligands. However, several findings (Dubois *et al.*, 1997; Gu & Du, 1998; Ichimura *et al.*, 1995) suggest that additional interactions may occur on the outer surface of 14-3-3. This may confer isoform specificity, since residues that are variable between 14-3-3 isoforms are located on the surface of the protein. It is also conceivable that due to the common occurrence of two 14-3-3 binding sites within the target protein, the synergy between the two may also lead to isoform preference of interaction. Studies have shown that 14-3-3 isoforms form heterodimers *in vivo* (Alvarez *et al.*, 2003; Liang *et al.*, 2008). The isoform-specific interaction with client proteins may become most relevant in a 'scaffolding' model, where a hetero-dimer consisting of different isoforms would bind to two separate targets via each isoform. Thus, it is likely that its propensity to form homo- and various heterodimeric combinations is crucial for the specificity of 14-3-3 isoform functions. Many apparent conclusions of 14-3-3 function within particular cell types are based on observations of a single isoform, and comparative data among isoforms are still limited. More analysis of the exact combinations of homo- and hetero-dimers of 14-3-3 isoforms that are present within cell compartments and that are

Alvarez, D., Callejo, M., Shoucri, R., Boyer, L., Price, G.B. & Zannis-Hadjopoulos, M. (2003)

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on mammalian DNA replication in vitro. *Biochemistry*, 42, 7205-7215. Aslan, J.E., You, H., Williamson, D.M., Endig, J., Youker, R.T., Thomas, L., Shu, H., Du, Y.,

Analysis of the cruciform binding activity of recombinant 14-3-3zeta-MBP fusion protein, its heterodimerization profile with endogenous 14-3-3 isoforms, and effect

Milewski, R.L., Brush, M.H., Possemato, A., Sprott, K., Fu, H., Greis, K.D., Runckel, D.N., Vogel, A. & Thomas, G. (2009) Akt and 14-3-3 control a PACS-2 homeostatic switch that integrates membrane traffic with TRAIL-induced apoptosis. *Mol Cell*,

involved in interactions with particular proteins will be important.

different cellular compartments.

**4. References**

34, 497-509.

Furthermore, 14-3-3 also controls the surface expression level of ENaC by regulating its degradation machinery. Ubiquitination of ENaC by ubiquitin-E3 protein ligase Nedd4-2 leads to an increased rate of protein degradation. The activation of SGK1 by aldosterone results in the phosphorylation of Nedd4-2 and recruitment of 14-3-3 proteins to Nedd4-2. This 14-3-3 binding inhibits the interaction of Nedd4-2 with ENaC and thereby suppresses the ubiquitin-dependent degradation of ENaC (Ichimura *et al.*, 2005; Liang *et al.*, 2006). This results in the longer stability of ENaC on the cell surface.

14-3-3s also bind to another transport machinery protein, phosphofurin acidic cluster sorting protein (PACS)-2 (Aslan *et al.*, 2009). PACS-2 recognizes an acidic cluster in the cytoplasmic tail of TRPP2 cation channel and localize the channel in the ER through interaction with the COPI complex (Kottgen *et al.*, 2005). The 14-3-3 binding to PACS-2 is dependent on the phosphorylation of PACS-2 by AKt, and this 14-3-3 binding was found to be required for the ER targeting of the PACS-2 substrate cargo protein TRPP2 (Aslan *et al.*, 2009). How binding of PACS-2 to 14-3-3 and COPI cooperate to mediate cargo traffic remains to be determined.

Cell surface expression of membrane proteins could be also regulated by 14-3-3 through modulation of endocytic processes. Recent study demonstrates the interaction of 14-3-3 with transferrin receptor trafficking protein (TTP) (Chiba *et al.*, 2009). TTP specifically promotes the internalization of transferrin receptor (TfR), but not other receptors such as epidermal growth factor receptor (EGFR) and low-density lipoprotein receptor (LDLR), through the clathrin-dependent pathway (Tosoni *et al.*, 2005). 14-3-3 proteins directly bind to TTP in the Akt-dependent manner and this interaction was enhanced by oxidative stress (Chiba *et al.*, 2009). Although the *in vivo* role of this 14-3-3 binding in TTP transport was not shown in this study, it suggests another possible mechanism where kinase signaling utilizes 14-3-3 as an effector molecule to control the cell surface density of proteins.

#### **3. Conclusion**

The requirement of phosphorylation for 14-3-3 binding confers 14-3-3 proteins a primary role in regulating protein-protein interactions that are under the control of specific kinases and phosphatases. Accordingly, 14-3-3 constitutes a key player which stands at a point of cross-talk between a plethora of vital biological processes including signaling, metabolism, cell cycle, and protein trafficking. Although available data from biochemical, structural, and bioinformatics studies have provided substantial amounts of information that characterize 14-3-3/client interaction, several fundamental questions regarding 14-3-3 protein biology remain to be addressed.

Although proteomics have revealed over 200 proteins forming complex with 14-3-3, the information regarding kinases and phosphatases that regulate these interactions are limited. The relevant kinase is not known for the majority of 14-3-3-binding proteins and the substantial overlap between the recognition sites of numerous basophilic serine/threonine kinases and 14-3-3 binding sites make their identification very difficult. Equally important but even less well studied are the phosphatases relevant for 14-3-3 binding. When and how the 14-3-3 proteins dissociate from the client proteins is very poorly understood. Does it require dephosphorylation of the 14-3-3 binding site by phosphatases? If so, how can the phosphatases do that when the phosphorylated serine/threonine of a target protein is buried in the amphipathic ligand-binding groove of 14-3-3? Alternatively, does some other signaling event facilitate the release of 14-3-3s from client proteins? In the context of protein trafficking, 14-3-3 is known to modulate sorting of the client proteins by various different mechanisms. In many of the cases where surface membrane transport is promoted by 14-3-3 binding, 14-3-3 seems to impinge on the early step of ER-to-Golgi trafficking. However, except for a very small number of studies (Godde *et al.*, 2006; Okamoto & Shikano, 2011), it is not understood if 14-3-3 proteins remain bound to the client all along the trip to the cell surface or dissociate in any particular step of the vesicular trafficking by phosphatase activity. These problems make us realize that we still do not know enough about the biology of kinases and phosphatases, especially the spatio-temporal regulation of their activities in different cellular compartments.

Another important question that has been long discussed but not fully addressed is whether and how 14-3-3 isoforms play specific roles. The complete sequence conservation in the observed ligand-binding regions of 14-3-3 would support the hypothesis that there may be little isoform specificity in the interaction between 14-3-3 and client proteins; therefore isoform-specific function of 14-3-3 may result either from subcellular localization (Paul *et al.*, 2005; van Hemert *et al.*, 2004) or transcriptional regulation (Liang *et al.*, 2006) of particular isotypes rather than from inherent differences in their ability to bind to particular ligands. However, several findings (Dubois *et al.*, 1997; Gu & Du, 1998; Ichimura *et al.*, 1995) suggest that additional interactions may occur on the outer surface of 14-3-3. This may confer isoform specificity, since residues that are variable between 14-3-3 isoforms are located on the surface of the protein. It is also conceivable that due to the common occurrence of two 14-3-3 binding sites within the target protein, the synergy between the two may also lead to isoform preference of interaction. Studies have shown that 14-3-3 isoforms form heterodimers *in vivo* (Alvarez *et al.*, 2003; Liang *et al.*, 2008). The isoform-specific interaction with client proteins may become most relevant in a 'scaffolding' model, where a hetero-dimer consisting of different isoforms would bind to two separate targets via each isoform. Thus, it is likely that its propensity to form homo- and various heterodimeric combinations is crucial for the specificity of 14-3-3 isoform functions. Many apparent conclusions of 14-3-3 function within particular cell types are based on observations of a single isoform, and comparative data among isoforms are still limited. More analysis of the exact combinations of homo- and hetero-dimers of 14-3-3 isoforms that are present within cell compartments and that are involved in interactions with particular proteins will be important.

#### **4. References**

178 Protein Kinases

Furthermore, 14-3-3 also controls the surface expression level of ENaC by regulating its degradation machinery. Ubiquitination of ENaC by ubiquitin-E3 protein ligase Nedd4-2 leads to an increased rate of protein degradation. The activation of SGK1 by aldosterone results in the phosphorylation of Nedd4-2 and recruitment of 14-3-3 proteins to Nedd4-2. This 14-3-3 binding inhibits the interaction of Nedd4-2 with ENaC and thereby suppresses the ubiquitin-dependent degradation of ENaC (Ichimura *et al.*, 2005; Liang *et al.*, 2006). This

14-3-3s also bind to another transport machinery protein, phosphofurin acidic cluster sorting protein (PACS)-2 (Aslan *et al.*, 2009). PACS-2 recognizes an acidic cluster in the cytoplasmic tail of TRPP2 cation channel and localize the channel in the ER through interaction with the COPI complex (Kottgen *et al.*, 2005). The 14-3-3 binding to PACS-2 is dependent on the phosphorylation of PACS-2 by AKt, and this 14-3-3 binding was found to be required for the ER targeting of the PACS-2 substrate cargo protein TRPP2 (Aslan *et al.*, 2009). How binding of PACS-2 to 14-3-3 and COPI cooperate to mediate cargo traffic remains to be determined. Cell surface expression of membrane proteins could be also regulated by 14-3-3 through modulation of endocytic processes. Recent study demonstrates the interaction of 14-3-3 with transferrin receptor trafficking protein (TTP) (Chiba *et al.*, 2009). TTP specifically promotes the internalization of transferrin receptor (TfR), but not other receptors such as epidermal growth factor receptor (EGFR) and low-density lipoprotein receptor (LDLR), through the clathrin-dependent pathway (Tosoni *et al.*, 2005). 14-3-3 proteins directly bind to TTP in the Akt-dependent manner and this interaction was enhanced by oxidative stress (Chiba *et al.*, 2009). Although the *in vivo* role of this 14-3-3 binding in TTP transport was not shown in this study, it suggests another possible mechanism where kinase signaling utilizes 14-3-3 as an

The requirement of phosphorylation for 14-3-3 binding confers 14-3-3 proteins a primary role in regulating protein-protein interactions that are under the control of specific kinases and phosphatases. Accordingly, 14-3-3 constitutes a key player which stands at a point of cross-talk between a plethora of vital biological processes including signaling, metabolism, cell cycle, and protein trafficking. Although available data from biochemical, structural, and bioinformatics studies have provided substantial amounts of information that characterize 14-3-3/client interaction, several fundamental questions regarding 14-3-3 protein biology

Although proteomics have revealed over 200 proteins forming complex with 14-3-3, the information regarding kinases and phosphatases that regulate these interactions are limited. The relevant kinase is not known for the majority of 14-3-3-binding proteins and the substantial overlap between the recognition sites of numerous basophilic serine/threonine kinases and 14-3-3 binding sites make their identification very difficult. Equally important but even less well studied are the phosphatases relevant for 14-3-3 binding. When and how the 14-3-3 proteins dissociate from the client proteins is very poorly understood. Does it require dephosphorylation of the 14-3-3 binding site by phosphatases? If so, how can the phosphatases do that when the phosphorylated serine/threonine of a target protein is buried in the amphipathic ligand-binding groove of 14-3-3? Alternatively, does some other

results in the longer stability of ENaC on the cell surface.

effector molecule to control the cell surface density of proteins.

**3. Conclusion**

remain to be addressed.

Phosphorylation-Regulated Cell Surface Expression of Membrane Proteins 181

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**8**

*Japan*

**Regulation of Retrotransposition**

*2Division of Animal Model, Department of Infections Diseases,*

**by Mitogen-Activated Protein Kinases**

Yukihito Ishizaka1,\*, Noriyuki Okudaira1 and Tadashi Okamura2

Our genome contains a higher amount of endogenous retroelements (〜42 %) than mouse ( 〜37 %) or fruit fly (〜3.6 %) (1-3). Long interspersed element-1 (L1) is the most abundant of transposable elements, comprising 〜17% of the genome (1-4). L1 is an autonomous endogenous retroelement that has evolved in a single, unbroken lineage for the past 40 million years in primates (5). A single human cell has more than 5x105 copies of L1 (2,4), and most of them are functionally defective (6). However, 80 to 100 copies of L1 are competent for retrotransposition (L1-RTP) (7), and approximately 10 % of these are highly active for "copy and paste" (7). L1 is actively expressed in embryonal stem cells (8) and L1-RTP is induced in oocytes or early embryonic development (9-11). L1-RTP occurring in germ lines would function an intrinsic factor responsible for allelic variants among individuals (12,13). However, aberrant L1-RTP alternates critical gene structures, leading to the development of inborn errors (14). At the moment, at least 17 genetic diseases have been reported as sporadic cases of inheritable disorders caused by aberrant insertion of L1 (14). On the other hand, recent observations suggest that L1-RTP occurs in somatic cells. Strikingly, it was shown that copy numbers of L1 is increased in human brain tissues (15,16). Aberrant L1 insertions have been detected in c-*myc* gene and the *APC* gene in breast carcinoma and colon carcinoma, respectively (17,18). Moreover recent analysis demonstrated that L1 is frequently mobilized in human lung cancers and pancreatic carcinomas (19,20). These observations indicate that it is important to understand the mode of L1-RTP, but little is known about the cellular factors for the induction of L1-RTP in somatic cells. We herein summarize our current understanding of L1-RTP induction, with an emphasis on mitogen-activated protein kinases (MAPKs), which are activated by environmental compounds, and we discuss their

**1. Introduction**

roles in genome shuffling.

\* Corresponding Author

**of Long Interspersed Element-1**

*1Department of Intractable Diseases,*

*National Center for Global Health and Medicine,*

*National Center for Global Health and Medicine*

## **Regulation of Retrotransposition of Long Interspersed Element-1 by Mitogen-Activated Protein Kinases**

Yukihito Ishizaka1,\*, Noriyuki Okudaira1 and Tadashi Okamura2

*1Department of Intractable Diseases, National Center for Global Health and Medicine, 2Division of Animal Model, Department of Infections Diseases, National Center for Global Health and Medicine Japan*

#### **1. Introduction**

186 Protein Kinases

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22, 537-548.

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BCL-X(L). *Cell*, 87, 619-628.

Our genome contains a higher amount of endogenous retroelements (〜42 %) than mouse ( 〜37 %) or fruit fly (〜3.6 %) (1-3). Long interspersed element-1 (L1) is the most abundant of transposable elements, comprising 〜17% of the genome (1-4). L1 is an autonomous endogenous retroelement that has evolved in a single, unbroken lineage for the past 40 million years in primates (5). A single human cell has more than 5x105 copies of L1 (2,4), and most of them are functionally defective (6). However, 80 to 100 copies of L1 are competent for retrotransposition (L1-RTP) (7), and approximately 10 % of these are highly active for "copy and paste" (7). L1 is actively expressed in embryonal stem cells (8) and L1-RTP is induced in oocytes or early embryonic development (9-11). L1-RTP occurring in germ lines would function an intrinsic factor responsible for allelic variants among individuals (12,13). However, aberrant L1-RTP alternates critical gene structures, leading to the development of inborn errors (14). At the moment, at least 17 genetic diseases have been reported as sporadic cases of inheritable disorders caused by aberrant insertion of L1 (14). On the other hand, recent observations suggest that L1-RTP occurs in somatic cells. Strikingly, it was shown that copy numbers of L1 is increased in human brain tissues (15,16). Aberrant L1 insertions have been detected in c-*myc* gene and the *APC* gene in breast carcinoma and colon carcinoma, respectively (17,18). Moreover recent analysis demonstrated that L1 is frequently mobilized in human lung cancers and pancreatic carcinomas (19,20). These observations indicate that it is important to understand the mode of L1-RTP, but little is known about the cellular factors for the induction of L1-RTP in somatic cells. We herein summarize our current understanding of L1-RTP induction, with an emphasis on mitogen-activated protein kinases (MAPKs), which are activated by environmental compounds, and we discuss their roles in genome shuffling.

<sup>\*</sup> Corresponding Author

Regulation of Retrotransposition

induced L1-RTP has yet to be identified.

of TPA-dependent tumor promotion.

**5. MAPKs required for L1-RTP by FICZ**

of Long Interspersed Element-1 by Mitogen-Activated Protein Kinases 189

AhR and ARNT1 are members of the basic helix-loop-helix/per-arnt-sim (bHLH/PAS) family, which are transcription factors involved in a variety of biological functions (41). Recently, it was shown that the bHLH/PAS family is functionally linked with environmental adaptation of living organisms (42). When AhR binds environmental compounds, it forms a heterodimer with ARNT1, which is recruited from the cytoplasm to chromatin and recognizes a xenobiotic responsive element (XRE) (36). It has been shown that the chromatin recruitment of ligand-bound AhR depends on the nuclear localization signal of ARNT1 (43), but there are no reports showing that ARNT1 functions as a receptor for environmental compounds. A cellular factor that cooperates with ARNT1 in FICZ-

To explore the involvement of MAPKs in L1-RTP, we extended our experiments to explore whether environmental carcinogens induce L1-RTP. In two-stage chemical carcinogenesis, it has been shown that skin tumors develop by treatment with 7,12-dimethylbenz[*a*]anthracene (DMBA) plus 12-*O*-tetradecanoylphorbol-13-acetate (TPA) (44). DMBA functions as an initiator and activates *H-ras* gene, whereas TPA functions as a tumor promoter thorough nongenotoxic effects (45). However, how TPA induces tumor progression remains to be clarified. We first analyzed whether L1-RTP is involved under skin carcinogenesis. When transgenic mice harboring human L1 as a transgene (hL1-EGFP mouse) were subjected to DMBA/TPAinduced skin carcinogenesis, L1-RTP was frequently observed in the DMBA/TPA-induced skin tumors (46). Interestingly, i*n vitro* experiments revealed that both DMBA and TPA were active for the induction of L1-RTP. On the other hand, *in vivo* experiments, in which hL1-EGFP mice were transiently treated with DMBA or TPA suggested that L1-RTP in the skin tumors was attributable to the effects of the repeated treatment with TPA. Notably, we observed that the mode of L1-RTP by DMBA and TPA was different. DMBA-induced L1-RTP was dependent on both AhR and ARNT1, whereas TPA-induced L1-RTP required neither protein. Instead, it depended on ERK1/2 and epidermal growth factor receptor (EGFR). Since Balmain *et al* (44) originally reported on DMBA/TPA-induced two-stage carcinogenesis, a major issue of cancer research is to clarify the mechanism of the TPA-induced tumor promotion. Using genetically-engineered mice, it has been proven that TPA-induced tumor promotion depends on ERK1/2 and EGFR (47,48). Interestingly, TPA-induced L1-RTP was shown to be dependent on these molecules, suggesting that the genome shuffling by L1-RTP is linked with the mode

**6. MAPKs are involved in the induction of L1-RTP by carcinogens**

Given that environmental compounds seemed to induce L1-RTP by involving different cellular proteins, we investigated other carcinogens such as B[*a*]P and 3-methylcholanthrene (3-MC). Consistent with a previous report (35), B[*a*]P induced L1-RTP in an AhR-dependent manner (46). Additionally, 3-MC also induced L1-RTP in an AhR-dependent manner (46). However, we found that the L1-RTP was induced even when siRNA against *ARNT1* was transfected into the cell (Fig. 1b, lanes 9 and 18). The siRNA clearly suppressed the mRNA expression of *CYP1A1* (Fig. 1c, lanes 11 and 12), indicating that the siRNA effectively abrogated the function of endogenous ARNT1 protein. These data support the idea that ARNT1 is dispensable for the induction of L1-RTP by these compounds. Because it has been

#### **2. Biology of L1-RTP**

L1, a non-long terminal repeat (non-LTR)-type endogenous retroelement, encodes two proteins: open reading frames 1 and 2 (ORF1 and 2) (3). ORF1 is a cytoplasmic 40 kDa protein that is present within ribonucleoprotein complexes (21-23). ORF1 associates in *cis* with L1-mRNA (24) and functions as a chaperone of L1-mRNA (25). ORF2 is a protein of about 150 kDa with dual activities as reverse transcriptase (RT) (26) and an endonuclease (27). ORF2 recognizes the 5′-TTAAAA hexanucleotide in the genome and induces a nick between 3′-AA and TTTT in the complementary strand (28,29). It has been proposed that the first-strand DNA is synthesized by target site-primed reverse transcription (3,29). ORF1 and 2 complete the entire process of L1-RTP and are competent for the induction of retrotransposition of *Alu*, a non-autonomous retroelements (30, 31).

### **3. Reported triggers of L1-RTP**

As to the environmental factors that induce L1-RTP in somatic cells, Farkash *et al*. reported that gamma irradiation at 4.5 Gy induced L1-RTP (32). Independently, Deiniger's group reported that heavy metals of such as mercury, cadmium and nickel also induced L1-RTP (33,34). They also reported that nickel-induced L1-RTP is induced by a post-transcriptional mechanism (34). As to an environmental carcinogen, Stribinskis and Ramos found that benzo[*a*]pyrene (B[*a*]P) induced L1-RTP (35). An extensive analysis revealed that aryl hydrocarbon receptor (AhR), which serves as a receptor for such environmental pollutants as 2,3,7,8-tetrachlorodibenzo-*p*-dioxin (TCDD) (36), was required for the B[*a*]P-induced L1- RTP (35). Because TCDD, a non-genotoxic hydrocarbon carcinogen, did not induce L1-RTP, it was proposed that as one of the its mechanisms an AhR-dependent cellular response converts B[*a*]P into an active genotoxic compound, which in turn induces L1-RTP (35). Although the exact modes of L1-RTP are unclear, these studies inspired us to investigate the possibility that various environmental compounds can induce L1-RTP.

#### **4. Induction of L1-RTP by an environmental compound and identification of p38 as a pivotal cellular factor**

First, we found that 6-formylindolo[3,2-*b*]carbazole (FICZ), a tryptophan photoproduct, induced L1-RTP (37). FICZ is highly active, and even picomolar comncentratiom of the compound induced L1-RTP. In mammalian cells, six groups of MAPKs, namely extracellular signal-regulated protein kinase (ERK)1/2, ERK5, JNK, p38, ERK3/4 and ERK7/8, are identified, and are activated by intracellular and extracellular stimuli (38). Among these, cellular signal cascades of ERK1/2, p38 and JNK have been well characterized, because of the availability of inhibitors, including PD98,059, SB202190 and SP600125, respectively. Using these MAPK inhibitors, we found that FICZ-induced L1-RTP was dependent on p38 (37). Interestingly, the compound induced phosphorylation of cyclic-AMP responsive element binding protein (CREB), and the down-regulation of endogenous CREB by short interference RNA (siRNA) attenuated the induction of L1-RTP by FICZ. Moreover, a transfection-back experiment of cDNA that encoded a siRNA-resistant CREB restored the induction of L1-RTP. These data indicate that the induction of L1-RTP by FICZ depended on p38-CREB-dependent signaling. Intriguingly, L1-RTP by FICZ was not dependent on AhR, although FICZ is a candidate physiological ligand of AhR (39). In contrast, L1-RTP by FICZ was dependent on AhR nuclear translocator 1 (ARNT1), a binding partner of AhR (40).

AhR and ARNT1 are members of the basic helix-loop-helix/per-arnt-sim (bHLH/PAS) family, which are transcription factors involved in a variety of biological functions (41). Recently, it was shown that the bHLH/PAS family is functionally linked with environmental adaptation of living organisms (42). When AhR binds environmental compounds, it forms a heterodimer with ARNT1, which is recruited from the cytoplasm to chromatin and recognizes a xenobiotic responsive element (XRE) (36). It has been shown that the chromatin recruitment of ligand-bound AhR depends on the nuclear localization signal of ARNT1 (43), but there are no reports showing that ARNT1 functions as a receptor for environmental compounds. A cellular factor that cooperates with ARNT1 in FICZinduced L1-RTP has yet to be identified.

### **5. MAPKs required for L1-RTP by FICZ**

188 Protein Kinases

L1, a non-long terminal repeat (non-LTR)-type endogenous retroelement, encodes two proteins: open reading frames 1 and 2 (ORF1 and 2) (3). ORF1 is a cytoplasmic 40 kDa protein that is present within ribonucleoprotein complexes (21-23). ORF1 associates in *cis* with L1-mRNA (24) and functions as a chaperone of L1-mRNA (25). ORF2 is a protein of about 150 kDa with dual activities as reverse transcriptase (RT) (26) and an endonuclease (27). ORF2 recognizes the 5′-TTAAAA hexanucleotide in the genome and induces a nick between 3′-AA and TTTT in the complementary strand (28,29). It has been proposed that the first-strand DNA is synthesized by target site-primed reverse transcription (3,29). ORF1 and 2 complete the entire process of L1-RTP and are competent for the induction of

As to the environmental factors that induce L1-RTP in somatic cells, Farkash *et al*. reported that gamma irradiation at 4.5 Gy induced L1-RTP (32). Independently, Deiniger's group reported that heavy metals of such as mercury, cadmium and nickel also induced L1-RTP (33,34). They also reported that nickel-induced L1-RTP is induced by a post-transcriptional mechanism (34). As to an environmental carcinogen, Stribinskis and Ramos found that benzo[*a*]pyrene (B[*a*]P) induced L1-RTP (35). An extensive analysis revealed that aryl hydrocarbon receptor (AhR), which serves as a receptor for such environmental pollutants as 2,3,7,8-tetrachlorodibenzo-*p*-dioxin (TCDD) (36), was required for the B[*a*]P-induced L1- RTP (35). Because TCDD, a non-genotoxic hydrocarbon carcinogen, did not induce L1-RTP, it was proposed that as one of the its mechanisms an AhR-dependent cellular response converts B[*a*]P into an active genotoxic compound, which in turn induces L1-RTP (35). Although the exact modes of L1-RTP are unclear, these studies inspired us to investigate the

**4. Induction of L1-RTP by an environmental compound and identification of**

First, we found that 6-formylindolo[3,2-*b*]carbazole (FICZ), a tryptophan photoproduct, induced L1-RTP (37). FICZ is highly active, and even picomolar comncentratiom of the compound induced L1-RTP. In mammalian cells, six groups of MAPKs, namely extracellular signal-regulated protein kinase (ERK)1/2, ERK5, JNK, p38, ERK3/4 and ERK7/8, are identified, and are activated by intracellular and extracellular stimuli (38). Among these, cellular signal cascades of ERK1/2, p38 and JNK have been well characterized, because of the availability of inhibitors, including PD98,059, SB202190 and SP600125, respectively. Using these MAPK inhibitors, we found that FICZ-induced L1-RTP was dependent on p38 (37). Interestingly, the compound induced phosphorylation of cyclic-AMP responsive element binding protein (CREB), and the down-regulation of endogenous CREB by short interference RNA (siRNA) attenuated the induction of L1-RTP by FICZ. Moreover, a transfection-back experiment of cDNA that encoded a siRNA-resistant CREB restored the induction of L1-RTP. These data indicate that the induction of L1-RTP by FICZ depended on p38-CREB-dependent signaling. Intriguingly, L1-RTP by FICZ was not dependent on AhR, although FICZ is a candidate physiological ligand of AhR (39). In contrast, L1-RTP by FICZ was dependent on AhR nuclear translocator 1 (ARNT1), a binding partner of AhR (40).

retrotransposition of *Alu*, a non-autonomous retroelements (30, 31).

possibility that various environmental compounds can induce L1-RTP.

**2. Biology of L1-RTP**

**3. Reported triggers of L1-RTP**

**p38 as a pivotal cellular factor**

To explore the involvement of MAPKs in L1-RTP, we extended our experiments to explore whether environmental carcinogens induce L1-RTP. In two-stage chemical carcinogenesis, it has been shown that skin tumors develop by treatment with 7,12-dimethylbenz[*a*]anthracene (DMBA) plus 12-*O*-tetradecanoylphorbol-13-acetate (TPA) (44). DMBA functions as an initiator and activates *H-ras* gene, whereas TPA functions as a tumor promoter thorough nongenotoxic effects (45). However, how TPA induces tumor progression remains to be clarified. We first analyzed whether L1-RTP is involved under skin carcinogenesis. When transgenic mice harboring human L1 as a transgene (hL1-EGFP mouse) were subjected to DMBA/TPAinduced skin carcinogenesis, L1-RTP was frequently observed in the DMBA/TPA-induced skin tumors (46). Interestingly, i*n vitro* experiments revealed that both DMBA and TPA were active for the induction of L1-RTP. On the other hand, *in vivo* experiments, in which hL1-EGFP mice were transiently treated with DMBA or TPA suggested that L1-RTP in the skin tumors was attributable to the effects of the repeated treatment with TPA. Notably, we observed that the mode of L1-RTP by DMBA and TPA was different. DMBA-induced L1-RTP was dependent on both AhR and ARNT1, whereas TPA-induced L1-RTP required neither protein. Instead, it depended on ERK1/2 and epidermal growth factor receptor (EGFR). Since Balmain *et al* (44) originally reported on DMBA/TPA-induced two-stage carcinogenesis, a major issue of cancer research is to clarify the mechanism of the TPA-induced tumor promotion. Using genetically-engineered mice, it has been proven that TPA-induced tumor promotion depends on ERK1/2 and EGFR (47,48). Interestingly, TPA-induced L1-RTP was shown to be dependent on these molecules, suggesting that the genome shuffling by L1-RTP is linked with the mode of TPA-dependent tumor promotion.

#### **6. MAPKs are involved in the induction of L1-RTP by carcinogens**

Given that environmental compounds seemed to induce L1-RTP by involving different cellular proteins, we investigated other carcinogens such as B[*a*]P and 3-methylcholanthrene (3-MC). Consistent with a previous report (35), B[*a*]P induced L1-RTP in an AhR-dependent manner (46). Additionally, 3-MC also induced L1-RTP in an AhR-dependent manner (46). However, we found that the L1-RTP was induced even when siRNA against *ARNT1* was transfected into the cell (Fig. 1b, lanes 9 and 18). The siRNA clearly suppressed the mRNA expression of *CYP1A1* (Fig. 1c, lanes 11 and 12), indicating that the siRNA effectively abrogated the function of endogenous ARNT1 protein. These data support the idea that ARNT1 is dispensable for the induction of L1-RTP by these compounds. Because it has been

Regulation of Retrotransposition

Cellular factors

AhR ARNT1 ER

○, dependent; -, independent; N.T., not tested.

MAPK

of Long Interspersed Element-1 by Mitogen-Activated Protein Kinases 191

replated for treatment with the compounds. Two days after the addition of 3 M B[*a*]P or 1 M 3-MC, the cells were harvested and their DNA extracted. No cytotoxicity was caused by 3 M B[*a*]P or 1 M 3-MC (data not shown). For the PCR-based assay, a spliced form of *EGFP* cDNA (140 bp in length) was amplified by PCR with primers specific for the separated exons of *EGFP* cDNA. The amplified DNA was then loaded onto an agarose gel and detected after staining with SYBR Green. As an internal control, the same samples were used as templates for the amplification of -actin. **a**. Effects of *ARNT1* siRNA on the downregulation of endogenous ARNT1. Western blot analysis was performed on day 2 after the transfection of *ARNT1* siRNA. U, untreated; C, control siRNA; *ARNT1*, *ARNT1* siRNA. **b**. L1- RTP caused by B[*a*]P and 3-MC was independent of ARNT1. The PCR-based assay of the effects of *ARNT1* siRNA is shown. HuH-7 cells were transfected with pEF06R on day 0 and then selected from days 1-3. On day 3, the cells were trypsinized, replated, and further transfected with control (Cont.) or *ARNT1* siRNA. On day 4, the cells were again divided into three groups and treated with DMSO, 3-MC (left panel), or B[*a*]P (right panel). After two days, DNA was extracted and subjected to a PCR-based assay. The arrowhead indicates the PCRamplified band corresponding to the induction of L1-RTP. **c**. *ARNT1* siRNA effectively blocked the mRNA expression of *CYP1A1*, which was induced by the compounds. HuH-7 cells were first transfected with control or *ARNT1* siRNAs. On day 2 after transfection, the cells were trypsinized, replated, and treated with B[*a*]P or 3-MC. RT-PCR analysis was performed on day 2 after the addition of the compounds. **d**. ER is required for the induction of L1-RTP by B[*a*]P or 3-MC. In this experiment, MCF-7 cells from a human breast carcinoma cell line

Inducers FICZ B[*a*]P 3-MC DMBA TPA

> ○ - ○

siRNA on the induction of

siRNA

○ ○ -

were used. Using a similar experimental protocol, the effect of *ER*

L1-RTP was examined. As an internal control, -actin was amplified.

The induction of L1-RTP was examined by a PCR-based assay (see legend for Fig. 1).

Table 1. Summary of cellular factors required for L1-RTP by environmental compounds

involvement of ER in the induction of L1-RTP. Interestingly, the transfection of *ER*

shown that AhR forms a complex with estrogen receptor (ER we further tested the

attenuated L1-RTP induced by these compounds (Fig. 1d, lanes 5 and 6). In addition, we found that CREB was definitely phorphorylated (Fig. 2a, lane 4), and checked the effects of MAPK inhibitors on the induction of L1-RTP by 3-MC. As shown in Fig. 2b, SB202190 attenuated the induction of L1-RTP (lane 8), whereas SP600125 did not (lane 10). To further

○ - ○

SB202190 ○ ○ ○ - - SP600125 - ○ ○ - - PD98,059 N.T. N.T. N.T. - ○

Fig. 1. B[*a*]P and 3-MC induced L1-RTP depending on AhR and ER, but not on ARNT1. An L1-RTP assay was performed according to the procedures described (37,46). Briefly, HuH-7 cells from a human hepatoma cell line were transfected with pEF06R on day 0, then treated with 0.5 g/mL puromycin for two days (days 1-3). The cells were then trypsinized and

Fig. 1. B[*a*]P and 3-MC induced L1-RTP depending on AhR and ER, but not on ARNT1. An L1-RTP assay was performed according to the procedures described (37,46). Briefly, HuH-7 cells from a human hepatoma cell line were transfected with pEF06R on day 0, then treated with 0.5 g/mL puromycin for two days (days 1-3). The cells were then trypsinized and

replated for treatment with the compounds. Two days after the addition of 3 M B[*a*]P or 1 M 3-MC, the cells were harvested and their DNA extracted. No cytotoxicity was caused by 3 M B[*a*]P or 1 M 3-MC (data not shown). For the PCR-based assay, a spliced form of *EGFP* cDNA (140 bp in length) was amplified by PCR with primers specific for the separated exons of *EGFP* cDNA. The amplified DNA was then loaded onto an agarose gel and detected after staining with SYBR Green. As an internal control, the same samples were used as templates for the amplification of -actin. **a**. Effects of *ARNT1* siRNA on the downregulation of endogenous ARNT1. Western blot analysis was performed on day 2 after the transfection of *ARNT1* siRNA. U, untreated; C, control siRNA; *ARNT1*, *ARNT1* siRNA. **b**. L1- RTP caused by B[*a*]P and 3-MC was independent of ARNT1. The PCR-based assay of the effects of *ARNT1* siRNA is shown. HuH-7 cells were transfected with pEF06R on day 0 and then selected from days 1-3. On day 3, the cells were trypsinized, replated, and further transfected with control (Cont.) or *ARNT1* siRNA. On day 4, the cells were again divided into three groups and treated with DMSO, 3-MC (left panel), or B[*a*]P (right panel). After two days, DNA was extracted and subjected to a PCR-based assay. The arrowhead indicates the PCRamplified band corresponding to the induction of L1-RTP. **c**. *ARNT1* siRNA effectively blocked the mRNA expression of *CYP1A1*, which was induced by the compounds. HuH-7 cells were first transfected with control or *ARNT1* siRNAs. On day 2 after transfection, the cells were trypsinized, replated, and treated with B[*a*]P or 3-MC. RT-PCR analysis was performed on day 2 after the addition of the compounds. **d**. ER is required for the induction of L1-RTP by B[*a*]P or 3-MC. In this experiment, MCF-7 cells from a human breast carcinoma cell line were used. Using a similar experimental protocol, the effect of *ER* siRNA on the induction of L1-RTP was examined. As an internal control, -actin was amplified.

○, dependent; -, independent; N.T., not tested.

The induction of L1-RTP was examined by a PCR-based assay (see legend for Fig. 1).

Table 1. Summary of cellular factors required for L1-RTP by environmental compounds

shown that AhR forms a complex with estrogen receptor (ER we further tested the involvement of ER in the induction of L1-RTP. Interestingly, the transfection of *ER*siRNA attenuated L1-RTP induced by these compounds (Fig. 1d, lanes 5 and 6). In addition, we found that CREB was definitely phorphorylated (Fig. 2a, lane 4), and checked the effects of MAPK inhibitors on the induction of L1-RTP by 3-MC. As shown in Fig. 2b, SB202190 attenuated the induction of L1-RTP (lane 8), whereas SP600125 did not (lane 10). To further

Regulation of Retrotransposition

of Long Interspersed Element-1 by Mitogen-Activated Protein Kinases 193

Fig. 2. MAPK is required for the induction of L1-RTP by B[*a*]P or 3-MC. **a**. Phosphorylation of MAPK substrates induced by 3-MC. HuH-7 cells were analyzed on day 2 after the addition of the compound. U, untreated; D, dimethylsulfoxide (DMSO); F, FICZ; M, 3-MC;

H, H2O2. **b**. Effects of MAPK inhibitors on L1-RTP induced by 3-MC. SB and SP are

identify a candidate substrate of p38, we examined the effects of *CREB* siRNA. The transfection of *CREB* siRNA abrogated the induction of L1-RTP by 3-MC (Fig. 2c). These data suggest that L1-RTP by 3-MC is induced by the cooperative function of AhR and ER depending on a signal cascade involving the p38-CREB pathway. Our data also indicate that the induction of L1-RTP by B[*a*]P is dependent on p38 and JNK (Fig. 2d, lanes 8 and 10).

Although further study is required, our current understanding is that various environmental compounds induce L1-RTP by combinations of the bHLH/PAS family and MAPKs (Table 1). L1-RTP was differentially induced by FICZ, DMBA, B[*a*]P, 3-MC and TPA. Most of the compounds examined, with the exception of DMBA, depended on MAPKs. Moreover, the L1-RTP by carcinogens depended on AhR, whereas FICZ did not. It is important to collect more information about chemical compounds active in the induction of L1-RTP and to elucidate the involvement of MAPKs.

It has been proposed that L1-RTP is controlled at the transcriptional and post-transcriptional levels. *In vitro* experiments revealed that that the expression of L1 is tightly regulated by methytlation of CpG in the region of 5'-LTR. In normal somatic cells, the 5'-LTR of L1 is methylated at CpG (50,51), but it is hypomethylated in transformed cells (52). It has been consistently reported that treatment with B[*a*]P induced the hypomethylation of CpG in HeLa cells (53). Moreover, it was reported that L1-5'UTR has a ubiquitously active antisense promoter that encodes small interfering RNAs, which effectively suppressed the retrotransposition of L1 (54). These observations indicate that epigenetic alternation of the 5'-UTR was proposed as the activation mode of L1-UTR by the compound. However, the following *in vitro* experiments suggested the presence of another regulatory system of L1- RTP. A reporter construct was transfected into cultured cells, and treatment with the compounds increased the frequency of L1-RTP. Because the reporter construct (*e.g.*, pEF06R, which carries *EGFP* cDNA as a reporter gene) contained a potent CMV promoter (32,34), L1 mRNA was strongly expressed when it was transfected into cultured cells. Even under such conditions, remarkable effects on the induction of L1-RTP were detectable by additing inducers such as FICZ, B[*a*]P, and 3-MC (37,46). Data indicate the presence of an additional regulatory system in which cellular proteins regulate the induction of L1-RTP. One possible mode of regulation is the chromatin recruitment of ORF1.

#### **7. The chromatin recruitment of ORF1 is MAPK-dependent**

Because it has been postulated that ORF1 is present in the cytoplasm (21-23) and carcinogeninduced L1-RTP was dependent on AhR, it is plausible that ORF1 is functionally associated with the bHLH/PAS family. To prove this, we evaluated the association of ORF1 and AhR by an immunoprecipitation followed by Western blot analysis with a polyclonal antibody to human ORF1. Intriguingly, ORF1 and AhR were associated even under normal conditions (Okudaira N, submitted). More importantly, we detected that recruitment of ORF1 into the chromatin-rich fraction was coupled with L1-RTP. As reported, chromatin recruitment of ORF1 was induced by FICZ in a MAPK-dependent manner. It is interesting that the chromatin recruitment of ORF1 was induced by FICZ, although FICZ-induced L1-RTP was not dependent on AhR (37). Interestingly, ARNT1 was associated with ORF1 when FICZ was added to the culture medium (37). Although the precise role of the MAPK is unclear, these data suggest that the chromatin recruitment of ORF1 is the important regulatory step in L1-RTP, where at least p38 is involved as a crucial cellular factor.

identify a candidate substrate of p38, we examined the effects of *CREB* siRNA. The transfection of *CREB* siRNA abrogated the induction of L1-RTP by 3-MC (Fig. 2c). These data suggest that L1-RTP by 3-MC is induced by the cooperative function of AhR and ER depending on a signal cascade involving the p38-CREB pathway. Our data also indicate that the induction of L1-RTP by B[*a*]P is dependent on p38 and JNK (Fig. 2d, lanes 8 and 10).

Although further study is required, our current understanding is that various environmental compounds induce L1-RTP by combinations of the bHLH/PAS family and MAPKs (Table 1). L1-RTP was differentially induced by FICZ, DMBA, B[*a*]P, 3-MC and TPA. Most of the compounds examined, with the exception of DMBA, depended on MAPKs. Moreover, the L1-RTP by carcinogens depended on AhR, whereas FICZ did not. It is important to collect more information about chemical compounds active in the induction of L1-RTP and to

It has been proposed that L1-RTP is controlled at the transcriptional and post-transcriptional levels. *In vitro* experiments revealed that that the expression of L1 is tightly regulated by methytlation of CpG in the region of 5'-LTR. In normal somatic cells, the 5'-LTR of L1 is methylated at CpG (50,51), but it is hypomethylated in transformed cells (52). It has been consistently reported that treatment with B[*a*]P induced the hypomethylation of CpG in HeLa cells (53). Moreover, it was reported that L1-5'UTR has a ubiquitously active antisense promoter that encodes small interfering RNAs, which effectively suppressed the retrotransposition of L1 (54). These observations indicate that epigenetic alternation of the 5'-UTR was proposed as the activation mode of L1-UTR by the compound. However, the following *in vitro* experiments suggested the presence of another regulatory system of L1- RTP. A reporter construct was transfected into cultured cells, and treatment with the compounds increased the frequency of L1-RTP. Because the reporter construct (*e.g.*, pEF06R, which carries *EGFP* cDNA as a reporter gene) contained a potent CMV promoter (32,34), L1 mRNA was strongly expressed when it was transfected into cultured cells. Even under such conditions, remarkable effects on the induction of L1-RTP were detectable by additing inducers such as FICZ, B[*a*]P, and 3-MC (37,46). Data indicate the presence of an additional regulatory system in which cellular proteins regulate the induction of L1-RTP. One possible

elucidate the involvement of MAPKs.

mode of regulation is the chromatin recruitment of ORF1.

**7. The chromatin recruitment of ORF1 is MAPK-dependent**

in L1-RTP, where at least p38 is involved as a crucial cellular factor.

Because it has been postulated that ORF1 is present in the cytoplasm (21-23) and carcinogeninduced L1-RTP was dependent on AhR, it is plausible that ORF1 is functionally associated with the bHLH/PAS family. To prove this, we evaluated the association of ORF1 and AhR by an immunoprecipitation followed by Western blot analysis with a polyclonal antibody to human ORF1. Intriguingly, ORF1 and AhR were associated even under normal conditions (Okudaira N, submitted). More importantly, we detected that recruitment of ORF1 into the chromatin-rich fraction was coupled with L1-RTP. As reported, chromatin recruitment of ORF1 was induced by FICZ in a MAPK-dependent manner. It is interesting that the chromatin recruitment of ORF1 was induced by FICZ, although FICZ-induced L1-RTP was not dependent on AhR (37). Interestingly, ARNT1 was associated with ORF1 when FICZ was added to the culture medium (37). Although the precise role of the MAPK is unclear, these data suggest that the chromatin recruitment of ORF1 is the important regulatory step

Fig. 2. MAPK is required for the induction of L1-RTP by B[*a*]P or 3-MC. **a**. Phosphorylation of MAPK substrates induced by 3-MC. HuH-7 cells were analyzed on day 2 after the addition of the compound. U, untreated; D, dimethylsulfoxide (DMSO); F, FICZ; M, 3-MC; H, H2O2. **b**. Effects of MAPK inhibitors on L1-RTP induced by 3-MC. SB and SP are

Regulation of Retrotransposition

**9. Further implications**

in L1-RTP, we focused on MAPKs in the current study.

genomes in the vicinity of the *cis*-element.

**10. Acknowledgments**

**11. References**

1626-32.

via phosphorylation.

of Long Interspersed Element-1 by Mitogen-Activated Protein Kinases 195

(38), a plausible model is that p38 augments the blocking activity of C/EBP- on APOBEC3G

Ataxia telangiectasis mutated (ATM), a phosphoinositide 3-kinase, has a functional link with L1-RTP (16). In an intriguing recent observation, the copy number of L1 increased in the brain tissues of patients with ATM (16). L1-RTP is consistently increased in the brain tissue of *ATM*-knock out mouse. Although these observations suggest that ATM functions as a negative regulator of L1-RTP, Gasior *et al*. originally reported that ATM was required for the induction of L1-RTP (65). Because of controversial observations regarding the role of ATM

Recent observations revealed that genome shuffling by L1-RTP in human somatic cells is a source of interindividual genomic heterogeneity (12,13). In addition, independent research groups reported that L1-RTP is frequently induced in tumors (19,20), suggesting the involvement of L1-RTP in the development of carcinogenesis. Importantly, L1 proteins are active on the retrotransposition of *Alu* (30,31), a non-autonomous retoroelement. On the other hand, it has been shown that *Alu* induces genomic instability via non-allelic homologous recombination (66). Thus, it is important to understand the activation mechanisms of L1. Our current observations support the idea that the chromatin recruitment of ORF1, which is controlled by cooperative regulation by members of the bHLH/PAS family and MAPKs, is a critical step in the regulation of L1-RTP. If this is the case, L1-RTP induction in the genome is selectively determined by cellular factors. Because AhR is a transcription factor that recognizes specific nucleotide element (36), carcinogens possibly induce L1-RTP in the

As observed in the analysis of L1-RTP by B[*a*]P and 3-MC, L1-RTP was not induced via the classical pathway controlled by both AhR and ARNT1. Our data suggest that L1-RTP is not necessarily induced by genotoxic activities of these compounds, further implying that L1-RTP is a novel type of genomic instability by which cellular cascades activated by environmental compounds lead to genome shuffling and generate stable phenotypes of the affected cells. The suppression of L1-RTP in somatic cells by targeting MAPK activity may be a novel strategy to

We are grateful to Dr. Elena. T. Luning Prak (University of Pennsylvania Medical Center) for pEF06R. This work was supported in parts by a research grant for the Log-range Research Initiative (LRI) from Japan Chemical Industry Association (JCIA), and The Grant for National Center for Global Health and Medicine 21A-104) and a Grant-in-Aid for Research from the Ministry of Health, Labour, and Welfare of Japan (109156296). Mr. Noriyuki Okudaira is an applicant supported by Grant-in-Aid from the Tokyo Biochemical Research Foundation.

[1] Haig H Kazazian Jr, "Mobile elements: drivers of genome evolution", *Science* 303 (2004):

protect the development of intractable diseases that include carcinogenesis.

All authors declare that they have no conflict of interest for the current work.

SB202190 and SP600125, respectively. U, untreated; D, DMSO; F, FICZ; M, 3-MC; H, H2O2. Of note, L1-RTP caused by 3-MC was attenuated by SB (lane 8), but not by SP (lane 10). The arrowhead indicates the induction of L1-RTP. **c**. CREB is required for L1-RTP induced by 3- MC. Left panel: Western blot analysis detected efficient down-regulation of the endogenous protein by *CREB* siRNA. U, untreated; N, non-transfected; C, control siRNA; *CREB*, *CREB* siRNA. Right panel: PCR-based assay after the transfection of *CREB* siRNA. *CREB* siRNA attenuated L1-RTP induced by 3-MC (lane 9). **d**. Effects of MAPK inhibitors on L1-RTP induced by B[*a*]P. Reagents similar to those described in Fig. 2b were used. L1-RTP by B[*a*]P was attenuated by both SB (lane 8) and SP (lane 10).

#### **8. Roles of MAPK on L1-RTP**

It has been supposed that the increase of transposable elements coupled with evolution (7). Even in *Candida albicans*, an L1-like structure is present as a functional gene (55). On the other hand, the bHLH/PAS family, which has a variety of biological functions including the metabolism of xenobiotics, maintenance of the circadian rhythm, cellular responses to hypoxia, and neuronal differentiation (41,42), is also well conserved from lower species to mammals (56). Interestingly, *AhR* homologs are also present in the genomes of *Drosophila melanogaster* and *Caenorhabditis elegans* (56). Although no direct evidence on the functional relationship between these two biological phenomena has been claimed, our observation is the first to demonstrate the functional link of these biological events. Moreover, data suggest that MAPKs are involved in the bHLH/PAS-dependent L1-RTP. MAPKs are involved in cellular response to intracellular and extracellular stress (38,57), and it is plausible that MAPKs mediate various stresses in the induction of L1-RTP, resulting in genome shuffling. Random mutagenesis by L1-RTP may give emerging novel organisms to survive in altered environments.

It is important to clarify the roles of MAPKs in the induction of L1-RTP. At present, at least two functions of MAPKs can be postulated. As explained, environmental compounds activate MAPKs, by which the chromatin recruitment of ORF1 is induced as a necessary step in L1-RTP. ORF1 functions in *cis* with L1- mRNA and functions as a chaperon of L1-mRNA (24,25). Using MAPK inhibitors, we observed that L1-RTP was abrogated concomitantly with the reduced chromatin recruitment of ORF1. These observations suggest that MAPK activation drives the mobilization of ORF1 to chromatin, by which retroelements are translocated to chromtin.

Another possible role for MAPKs is related to the activity of the APOBEC family. It has been proposed that APOBEC family functions as innate restriction factors that suppress the activity of endogenous retroelements (58). Originally, it was postulated that the APOBEC family inhibits HIV-1 infection by editing C to T via deaminase activity (58). Vif, a gene product of HIV-1, degrades APOBEC proteins, causing infected cells to become permissive for HIV-1 infection (59). We previously showed that all members of the APOBEC family exhibit inhibitory activity toward L1-RTP (60). However, it was recently postulated that the APOBEC has dual activity (61) and inhibits the activity of RT (62). In *in vitro* experiments in which APOBEC3G were added to the reaction of RT in the synthesis of viral DNA, APOBEC interfered with elongation of the viral DNA (62). Interestingly, it has been shown that C/EBP- bound APOBEC3G and attenuate the inhibitory activity of APOBEC3G (63). Moreover, it was demonstrated that the mutation of serine at 228 (S228), the phosphorylation of which is correlated with the cytoplasmic localization of the molecule (63), abolished both binding and inhibitory activity on APOBEC3G (64). Given that C/EBP-is a substrate of p38 (38), a plausible model is that p38 augments the blocking activity of C/EBP- on APOBEC3G via phosphorylation.

### **9. Further implications**

194 Protein Kinases

SB202190 and SP600125, respectively. U, untreated; D, DMSO; F, FICZ; M, 3-MC; H, H2O2. Of note, L1-RTP caused by 3-MC was attenuated by SB (lane 8), but not by SP (lane 10). The arrowhead indicates the induction of L1-RTP. **c**. CREB is required for L1-RTP induced by 3- MC. Left panel: Western blot analysis detected efficient down-regulation of the endogenous protein by *CREB* siRNA. U, untreated; N, non-transfected; C, control siRNA; *CREB*, *CREB* siRNA. Right panel: PCR-based assay after the transfection of *CREB* siRNA. *CREB* siRNA attenuated L1-RTP induced by 3-MC (lane 9). **d**. Effects of MAPK inhibitors on L1-RTP induced by B[*a*]P. Reagents similar to those described in Fig. 2b were used. L1-RTP by B[*a*]P

It has been supposed that the increase of transposable elements coupled with evolution (7). Even in *Candida albicans*, an L1-like structure is present as a functional gene (55). On the other hand, the bHLH/PAS family, which has a variety of biological functions including the metabolism of xenobiotics, maintenance of the circadian rhythm, cellular responses to hypoxia, and neuronal differentiation (41,42), is also well conserved from lower species to mammals (56). Interestingly, *AhR* homologs are also present in the genomes of *Drosophila melanogaster* and *Caenorhabditis elegans* (56). Although no direct evidence on the functional relationship between these two biological phenomena has been claimed, our observation is the first to demonstrate the functional link of these biological events. Moreover, data suggest that MAPKs are involved in the bHLH/PAS-dependent L1-RTP. MAPKs are involved in cellular response to intracellular and extracellular stress (38,57), and it is plausible that MAPKs mediate various stresses in the induction of L1-RTP, resulting in genome shuffling. Random mutagenesis by

It is important to clarify the roles of MAPKs in the induction of L1-RTP. At present, at least two functions of MAPKs can be postulated. As explained, environmental compounds activate MAPKs, by which the chromatin recruitment of ORF1 is induced as a necessary step in L1-RTP. ORF1 functions in *cis* with L1- mRNA and functions as a chaperon of L1-mRNA (24,25). Using MAPK inhibitors, we observed that L1-RTP was abrogated concomitantly with the reduced chromatin recruitment of ORF1. These observations suggest that MAPK activation drives the mobilization of ORF1 to chromatin, by which retroelements are

Another possible role for MAPKs is related to the activity of the APOBEC family. It has been proposed that APOBEC family functions as innate restriction factors that suppress the activity of endogenous retroelements (58). Originally, it was postulated that the APOBEC family inhibits HIV-1 infection by editing C to T via deaminase activity (58). Vif, a gene product of HIV-1, degrades APOBEC proteins, causing infected cells to become permissive for HIV-1 infection (59). We previously showed that all members of the APOBEC family exhibit inhibitory activity toward L1-RTP (60). However, it was recently postulated that the APOBEC has dual activity (61) and inhibits the activity of RT (62). In *in vitro* experiments in which APOBEC3G were added to the reaction of RT in the synthesis of viral DNA, APOBEC interfered with elongation of the viral DNA (62). Interestingly, it has been shown that C/EBP- bound APOBEC3G and attenuate the inhibitory activity of APOBEC3G (63). Moreover, it was demonstrated that the mutation of serine at 228 (S228), the phosphorylation of which is correlated with the cytoplasmic localization of the molecule (63), abolished both binding and inhibitory activity on APOBEC3G (64). Given that C/EBP-is a substrate of p38

L1-RTP may give emerging novel organisms to survive in altered environments.

was attenuated by both SB (lane 8) and SP (lane 10).

**8. Roles of MAPK on L1-RTP**

translocated to chromtin.

Ataxia telangiectasis mutated (ATM), a phosphoinositide 3-kinase, has a functional link with L1-RTP (16). In an intriguing recent observation, the copy number of L1 increased in the brain tissues of patients with ATM (16). L1-RTP is consistently increased in the brain tissue of *ATM*-knock out mouse. Although these observations suggest that ATM functions as a negative regulator of L1-RTP, Gasior *et al*. originally reported that ATM was required for the induction of L1-RTP (65). Because of controversial observations regarding the role of ATM in L1-RTP, we focused on MAPKs in the current study.

Recent observations revealed that genome shuffling by L1-RTP in human somatic cells is a source of interindividual genomic heterogeneity (12,13). In addition, independent research groups reported that L1-RTP is frequently induced in tumors (19,20), suggesting the involvement of L1-RTP in the development of carcinogenesis. Importantly, L1 proteins are active on the retrotransposition of *Alu* (30,31), a non-autonomous retoroelement. On the other hand, it has been shown that *Alu* induces genomic instability via non-allelic homologous recombination (66). Thus, it is important to understand the activation mechanisms of L1. Our current observations support the idea that the chromatin recruitment of ORF1, which is controlled by cooperative regulation by members of the bHLH/PAS family and MAPKs, is a critical step in the regulation of L1-RTP. If this is the case, L1-RTP induction in the genome is selectively determined by cellular factors. Because AhR is a transcription factor that recognizes specific nucleotide element (36), carcinogens possibly induce L1-RTP in the genomes in the vicinity of the *cis*-element.

As observed in the analysis of L1-RTP by B[*a*]P and 3-MC, L1-RTP was not induced via the classical pathway controlled by both AhR and ARNT1. Our data suggest that L1-RTP is not necessarily induced by genotoxic activities of these compounds, further implying that L1-RTP is a novel type of genomic instability by which cellular cascades activated by environmental compounds lead to genome shuffling and generate stable phenotypes of the affected cells. The suppression of L1-RTP in somatic cells by targeting MAPK activity may be a novel strategy to protect the development of intractable diseases that include carcinogenesis.

#### **10. Acknowledgments**

We are grateful to Dr. Elena. T. Luning Prak (University of Pennsylvania Medical Center) for pEF06R. This work was supported in parts by a research grant for the Log-range Research Initiative (LRI) from Japan Chemical Industry Association (JCIA), and The Grant for National Center for Global Health and Medicine 21A-104) and a Grant-in-Aid for Research from the Ministry of Health, Labour, and Welfare of Japan (109156296). Mr. Noriyuki Okudaira is an applicant supported by Grant-in-Aid from the Tokyo Biochemical Research Foundation.

All authors declare that they have no conflict of interest for the current work.

#### **11. References**

[1] Haig H Kazazian Jr, "Mobile elements: drivers of genome evolution", *Science* 303 (2004): 1626-32.

Regulation of Retrotransposition

of Long Interspersed Element-1 by Mitogen-Activated Protein Kinases 197

[25] Sandra L Martin, "Nucleic acid chaperone properties of ORF1p from the non-LTR

[26] Qinghua Feng *et al*., "Human L1 retrotransposison encodes a conserved endonuclease

[27] Stephen ML Mathias e*t al.*, "Reverse transcriptase encoded by a human transposable

[28] Jerzy Jurka, "Sequence patterns indicate an enzymatic involvement in integration of

[29] Nicolas Gilbert *et al.*, "Multiple fates of L1 retrotransposition intermediates in cultured

[30] Marie Dewannieux, Cecile Esnault and Thierry Heidmann, "LINE-mediated retrotransposition of marked Alu sequences", *Nat Genet* 35 (2003): 41-8. [31] Nichola Wallace *et al*., "LINE-1 ORF1 protein enhances Alu SINE retrotrasposition",

[32] Evan A Farkash *et al*., "Gamma radiation increases endonuclease-dependent L1 retrotransposition in a cultured cell assay", *Nucleic Acids Res* 34 (2006): 1196-1204. [33]  "The aryl hydrocarbon receptor complex and the control of

[37] Noriyuki Okudaira *et al*., "Induction of long interspersed nucleotide element-1 (L1)

[38] Marie Cargnello and Philippe P Roux, "Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases", *Micobiol Mol Biol Rev* 75 (2011): 50-83. [39] Emma Wincent *et al*., "The suggested physiologic aryl hydrocarbon receptor activator

[40] Emilly C Hoffman *et al*., "Cloning of a factor required for activity of the Ah (dioxin)

[41] Robyn J Kewley *et al*., "The mammalian basic helix-loop-helix/PAS family of transcriptional regulators", *Int J Biochem Cell Biol* 36 (2004): 189-204. [42] Brian E McIntosh *et al.*, "Mammalian Per-Arnt-Sim proteins in environmental

[43] Hideaki Eguchi *et al*., "A nuclear localization signal of human aryl hydrocarbon

[44] Alian Balmain *et al.*, "Activation of the mouse cellular Harvey-ras gene in chemically

[45] Mark A Nelson *et al*., "Detection of mutant Ha-*ras* in chemically initiated mouse skin

induced benign skin papillomas", *Nature* 307 (1984): 658-60.

nuclear element 1 retrotransposition by benzo(*a*)pyrene, an ubiquitous

retrotransposition by 6-formylindolo[3,2-*b*]carbazole, a tryptophan photoproduct",

and cytochrome P4501 substrate 6-formylindolo[3,2-b]carbazole is present in

receptor nuclear translocator/hypoxia-inducible factor 1beta is a novel bipartite type recognized by the two components of nuclear pore-targeting complex", *J Biol*

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*3USA, 1,2,4Chile*

**Connexins as Substrates for Protein Kinases**

*1Departamento de Fisiología, Pontificia Universidad Católica de Chile, Santiago,*

José L. Vega1,2, Elizabeth Baeza1, Viviana M. Berthoud3 and Juan C. Sáez1,4

*3Department of Pediatrics, Section of Hematology, Oncology, University of Chicago, IL, 4Instituto Milenio, Centro Interdisciplinario de Neurociencias de Valparaíso, Valparaíso,*

Connexins are protein subunits expressed by cordates that form gap junction channels (GJCs) and hemichannels (HCs) (Goodenough, 1974; Makowski et al., 1977). A GJC is formed by the head-to-head docking of two HCs, each contributed by one of the two contacting cells (Meşe et al., 2007). Each HC is an oligomeric assembly of six identical (homomeric) or six different (heteromeric) Cx subunits (Sáez et al., 2005). GJCs and HCs subserve different functions; while GJCs communicate the cytoplasm of contacting cells, HCs provide a pathway for communication between the intracellular and extracellular compartments (Bruzzone and Dermietzel, 2006). Although both types of channels are permeable to ions and small molecules, GJCs and HCs composed of the same Cx subtype are likely to present differences in permeability and regulatory properties (Sáez et al., 2003;