Compared to the media controls, the DMSO controls did not differ in phosphorylation levels of these specific P-Tyr or P-Thr. While the intensity of the bands were similar for each cell line at each of the two time points, exceptions were noted for Tyr 877 and cell lines BT-474 and MDA MD 453, where phosphorylation is clearly increased from the 8 hour to the 48 hour time point. Similarly, P-Tyr 1222 also demonstrated increased intensity from 8 to 48 hours in cell line MDA-MB-453. Whether these findings suggest that the cells are not completely recruited into log phase growth at the 8-hour time point or cell-cell contact plays a role is unclear. The actin and HER2 bands strongly suggest equivalence of cell number applied to each lane at each of these time points.

## *3.2.3. Tyrosine and threonine phosphorylation levels in trastuzumab and lapatinib treated cells*

Modulation of HER2 Tyrosine/Threonine Phosphorylation and Cell Signalling 365

in the cell pellet fraction (figure 2). Because concentrates of cytoplasmic fractions and nuclear fractions did not demonstrate the presence of this 20 kD peptide segment, a Triton X-114 fraction of the cell membrane was assayed and found to have high levels of this product (Figure 2). These results suggest that this product is derived from the extra-cellular domain of HER2 by enzymatic processing and binds avidly to a cell membrane-anchored determinant or is internalized into a sub-membrane compartment, co-purifying with the membrane fraction, or that it is a membrane protein cross reactive with antibody to extracellular domain (AA 183-373). The significance of this finding is that this peptide appears to be inhibited by lapatinib treatment while the intact HER2 levels remain constant and expression of this protein is unaffected by trastuzumab treatments (Figure 2). Thus, this small molecular weight product may be related to the unphosphorylated state of HER2.

HER2 transmembrane activation signals promote a high level of selected intracellular domain P-Tyr sites, which are then recognized by specific cytoplasmic signal transducer molecules [10]. Thus, HER2 acts as an oncogenic unit transmitting cell survival, growth, and cell proliferation signals to critical intracellular pathways such as Mitogen-activated protein kinases (MAPKs), AKT and phoshatidylinositol 3-OH kinase (PI3K) pathways[11,12,29]. Here, we show that the relative level of specific P-Tyr is different for each HER2 ampliconpositive cell line examined, and thus, each oncogenic unit may be distinct in each tumour [13]r. Linkage of anti-HER2 treatment to the inhibition of these downstream pathways is well established, suggesting that these specific P-Tyr are critical mediators of the downstream signalling effects of the activated HER2 state and cell survival [7,10-12,29,30]. The molecular mechanism by which trastuzumab, a humanized IgG1, modulates the intracellular tyrosine kinase site from its extra-cellular binding site has remained unanswered. For example, cetuximab binds to, or near, the ligand binding site of EGFR thus acting as an antagonist [1,2,30-32]. Defining the molecular mechanism by which trastuzumab mediates its anti-HER2 effects could reveal a general non-ligand site-specific strategy for development of receptor inhibitory monoclonal antibodies in other receptor

The primary focus of this investigation was to determine the key molecular mechanism(s) governing trastuzumab's transmembrane modulation of downstream effects. Here, we show that trastuzumab binding does not appear to alter the net steady state levels of p185 HER2 protein as detected by all three of the anti-HER2 antibodies tested. Previous gene mRNA expression array findings demonstrate that no significant change in mRNA levels for HER2 or the other members of the ERBb family were induced by trastuzumab binding [33]. Taken together, these results strongly argue against an antibody-dependent alteration in natural turnover rate or internalization kinetics of HER2 [34]. We conclude that in the absence of demonstrable modulation of HER2 membrane mass on the cell surface, HER2 protein synthesis, and thus, alteration of kinetics of internalization or HER2 metabolism, net

**4. Discussion**

kinase systems.

No discernable change in phosphorylation of the P-Tyr or P-Thr was detected for trastuzumab-treated cells despite ongoing inhibition of cell growth at these time points. The unexpected lack of trastuzumab inhibition of tyrosine phosphorylation prompted experiments that replaced fetal calf serum (FCS) with human serum to determine if a component of FCS was responsible for inhibiting the full trastuzumab activity. Cells grown in media with 10% human serum from breast cancer patients or normal females, pre or postmenopausal, or male serum did not affect these findings (data not shown). Doses of trastuzumab up to 250 micrograms/ml did also did not change the patterns of phosphorylation. In contrast to the results demonstrating constant HER2 protein steady state levels, P-Tyr levels in lapatinib treated cells are markedly reduced (Tyr 1248) or absent (Tyr 687, 1116 and 1223), consistent with its role as a HER2 tyrosine kinase inhibitor (Figure 1). In addition, the P-Thr level is not affected by lapatinib exposure, demonstrating the tyrosine backbone specificity of lapatinib. As the lapatinib Western blots demonstrate, HER2 and actin protein levels are maintained through 48 hours, these results strongly suggests that changes in detected phosphorylation levels are due to kinase inhibition rather than the substantial in HER2 protein or cell death. We suggest that these early findings are related to the lapatinib mechanism of cell death that is apparent 2-3 days later.

## **3.3. MRNA expression patterns in trastuzumab treated breast cancer cells**

### *3.3.1. Detection of a small molecular weight HER2-like product*

Cell fractionation experiments were carried out to determine whether HER2 derived products with nuclear localization motifs could be identified by Western Blot analysis and to determine if trastuzumab or lapatinib altered their molecular processing mechanism. Differential generation of enzymatically derived nuclear localizing products could be responsible for trastuzumab mediated trans-membrane growth inhibitory effects. The results indicate that these techniques were not sensitive enough to detect, or were not of the correct immune-specificity to detect HER2 derived product in concentrates of nuclear or cytoplasmic cell fractions (data not shown).

However, using the anti-HER2 external domain antibody (AA 182-373), we detected a small molecular weight (20 kD) external domain-like HER2-derived product associated with the cell (Figure 2). This peptide segment is too far from the hydrophobic transmembrane segment to contain a transmembrane anchoring component to explain its association with the cell pellet, as it is too small (approximately 180 amino-acids in length) to extend from position 373 to the transmembrane domain. This peptide was not detected in supernatant or concentrates of supernatants. Cells exposed to acid conditions (pH 4.0, 0.5 M acetate, 0.15 N NaCl) to elute this 20 kD segment from the cell surface demonstrated that the 20 kD peptide could not be eluted under these conditions and was still found associated and concentrated in the cell pellet fraction (figure 2). Because concentrates of cytoplasmic fractions and nuclear fractions did not demonstrate the presence of this 20 kD peptide segment, a Triton X-114 fraction of the cell membrane was assayed and found to have high levels of this product (Figure 2). These results suggest that this product is derived from the extra-cellular domain of HER2 by enzymatic processing and binds avidly to a cell membrane-anchored determinant or is internalized into a sub-membrane compartment, co-purifying with the membrane fraction, or that it is a membrane protein cross reactive with antibody to extracellular domain (AA 183-373). The significance of this finding is that this peptide appears to be inhibited by lapatinib treatment while the intact HER2 levels remain constant and expression of this protein is unaffected by trastuzumab treatments (Figure 2). Thus, this small molecular weight product may be related to the unphosphorylated state of HER2.

#### **4. Discussion**

364 Protein Phosphorylation in Human Health

*cells*

*3.2.3. Tyrosine and threonine phosphorylation levels in trastuzumab and lapatinib treated*

No discernable change in phosphorylation of the P-Tyr or P-Thr was detected for trastuzumab-treated cells despite ongoing inhibition of cell growth at these time points. The unexpected lack of trastuzumab inhibition of tyrosine phosphorylation prompted experiments that replaced fetal calf serum (FCS) with human serum to determine if a component of FCS was responsible for inhibiting the full trastuzumab activity. Cells grown in media with 10% human serum from breast cancer patients or normal females, pre or postmenopausal, or male serum did not affect these findings (data not shown). Doses of trastuzumab up to 250 micrograms/ml did also did not change the patterns of phosphorylation. In contrast to the results demonstrating constant HER2 protein steady state levels, P-Tyr levels in lapatinib treated cells are markedly reduced (Tyr 1248) or absent (Tyr 687, 1116 and 1223), consistent with its role as a HER2 tyrosine kinase inhibitor (Figure 1). In addition, the P-Thr level is not affected by lapatinib exposure, demonstrating the tyrosine backbone specificity of lapatinib. As the lapatinib Western blots demonstrate, HER2 and actin protein levels are maintained through 48 hours, these results strongly suggests that changes in detected phosphorylation levels are due to kinase inhibition rather than the substantial in HER2 protein or cell death. We suggest that these early findings are related to

the lapatinib mechanism of cell death that is apparent 2-3 days later.

*3.3.1. Detection of a small molecular weight HER2-like product*

cytoplasmic cell fractions (data not shown).

**3.3. MRNA expression patterns in trastuzumab treated breast cancer cells**

Cell fractionation experiments were carried out to determine whether HER2 derived products with nuclear localization motifs could be identified by Western Blot analysis and to determine if trastuzumab or lapatinib altered their molecular processing mechanism. Differential generation of enzymatically derived nuclear localizing products could be responsible for trastuzumab mediated trans-membrane growth inhibitory effects. The results indicate that these techniques were not sensitive enough to detect, or were not of the correct immune-specificity to detect HER2 derived product in concentrates of nuclear or

However, using the anti-HER2 external domain antibody (AA 182-373), we detected a small molecular weight (20 kD) external domain-like HER2-derived product associated with the cell (Figure 2). This peptide segment is too far from the hydrophobic transmembrane segment to contain a transmembrane anchoring component to explain its association with the cell pellet, as it is too small (approximately 180 amino-acids in length) to extend from position 373 to the transmembrane domain. This peptide was not detected in supernatant or concentrates of supernatants. Cells exposed to acid conditions (pH 4.0, 0.5 M acetate, 0.15 N NaCl) to elute this 20 kD segment from the cell surface demonstrated that the 20 kD peptide could not be eluted under these conditions and was still found associated and concentrated HER2 transmembrane activation signals promote a high level of selected intracellular domain P-Tyr sites, which are then recognized by specific cytoplasmic signal transducer molecules [10]. Thus, HER2 acts as an oncogenic unit transmitting cell survival, growth, and cell proliferation signals to critical intracellular pathways such as Mitogen-activated protein kinases (MAPKs), AKT and phoshatidylinositol 3-OH kinase (PI3K) pathways[11,12,29]. Here, we show that the relative level of specific P-Tyr is different for each HER2 ampliconpositive cell line examined, and thus, each oncogenic unit may be distinct in each tumour [13]r. Linkage of anti-HER2 treatment to the inhibition of these downstream pathways is well established, suggesting that these specific P-Tyr are critical mediators of the downstream signalling effects of the activated HER2 state and cell survival [7,10-12,29,30]. The molecular mechanism by which trastuzumab, a humanized IgG1, modulates the intracellular tyrosine kinase site from its extra-cellular binding site has remained unanswered. For example, cetuximab binds to, or near, the ligand binding site of EGFR thus acting as an antagonist [1,2,30-32]. Defining the molecular mechanism by which trastuzumab mediates its anti-HER2 effects could reveal a general non-ligand site-specific strategy for development of receptor inhibitory monoclonal antibodies in other receptor kinase systems.

The primary focus of this investigation was to determine the key molecular mechanism(s) governing trastuzumab's transmembrane modulation of downstream effects. Here, we show that trastuzumab binding does not appear to alter the net steady state levels of p185 HER2 protein as detected by all three of the anti-HER2 antibodies tested. Previous gene mRNA expression array findings demonstrate that no significant change in mRNA levels for HER2 or the other members of the ERBb family were induced by trastuzumab binding [33]. Taken together, these results strongly argue against an antibody-dependent alteration in natural turnover rate or internalization kinetics of HER2 [34]. We conclude that in the absence of demonstrable modulation of HER2 membrane mass on the cell surface, HER2 protein synthesis, and thus, alteration of kinetics of internalization or HER2 metabolism, net membrane HER2 levels could not be implicated as a mechanism to down-regulate HER2 signalling. Despite these observations for HER2, our gene mRNA expression array data did suggest numerous intracellular pathway modulations mediated by trastuzumab binding to the target cells examined here [33]. These findings confirm transmembrane signal transduction modulation directed and specific to trastuzumab binding [33-35]. For example, we found that, the ubiquitin pathway is up-regulated upon trastuzumab binding to HER2 [35], as has been reported by others through ligase activity at tyrosine 1112 [35,36].

Modulation of HER2 Tyrosine/Threonine Phosphorylation and Cell Signalling 367

*vitro* experiments may mislead investigators into attributing findings to cross-talk between receptor pathways rather than cross-reactivity of the reagent tested. We may over estimate biologic activity when reagents are used at too high of a concentration or too long of an exposure. Results of these experiments may mislead investigators into attributing findings to cross-talk between receptor pathways rather than the cross reactivity of the reagent tested. Thus, we conclude that results of experiments testing cetuximab's biologic effects more closely represent, and are more specific to, the pure anti-EGFR blockade. We clearly show here, in agreement with previous reports by others that pure anti-EGFR1 inhibition of signalling does not result in cell death or modulation of HER2 phosphorylation levels

These observations suggest that the anti-EGFR1 tyrosine kinase effects of lapatinib are not contributing to its anti-HER2 effects. Thus, the results of the panel of anti-HER2-P-Tyrantibodies demonstrating almost complete abrogation of HER2 phosphorylation by lapatinib and its associated delayed cell cytotoxicity (day 5-6) establishes a specific and unique link between HER2 tyrosine phosphorylation and cell survival. Importantly, these results also suggest that additional signalling through HER3 and HER4 may not be critical for apoptosis [40], except as it may ultimately be mediated through HER2 tyrosine kinase inhibition (reduction in hetero-dimerization) [40]. Thus, blocking dimerization by itself, which is unlikely to affect steric change in the intracellular domain, will not induce a modulation of P-Tyr levels. Taken together, these data suggest that testing the effect of a

In the clinic, where the anti-EGFR toxicity of lapatinib is dose limiting (diarrhea and skin rash) and prohibits dose escalation, the testing of a small molecule TKI manifesting pure HER2 kinase inhibition would be of great interest. Recently a pan ErbB TKI was tested and shown to be dose limited by the EGFR-mediated bowel toxicity, further supporting the conclusions presented here [41]. A pure anti-HER2 agent would be even more compelling if one could demonstrate upon dose escalation increased tumor cytotoxicity and a lack of other intervening non-HER2 related dose-limiting toxicities. Close examination of many of the TKIs demonstrate widespread promiscuity with many unexpected cross-reactivities [42]. In general, clinical success of a widely reactive kinase inhibitor is not informative as an identifier of the key pathway(s) responsible for mediating anti-tumor effects and often leads

Using the four anti-P-Tyr sera, we could not find any diminution in the phosphorylation levels as assayed by Western blots, in cells treated up to nine days in the presence of trastuzumab. In addition, the anti-phospho-threonine reagent was used as a negative control, and was found not to be blocked by lapatinib demonstrating the specificity of the drug and the kinase activity. Thus, we propose that the kinase inhibition that was originally described by the mouse antibody 4D5 [23] has been lost in the humanization process [25]. The abundance of well-documented evidence that inhibition of specific downstream

pathways is a consequence of trastuzumab-cell binding remains unexplained.

specific and robust HER2 tyrosine kinase inhibitory reagent is warranted.

to "assumed" mechanisms of activity.

[29].

Trastuzumab has not been definitively shown to retain the tyrosine kinase inhibitory activity of the parent 4D5 mouse monoclonal antibody [23]. Here we show that the level of HER2-P-Tyr/Thr corrected to cell number, actin levels, and the steady-state levels of intact extractable HER2, is not altered by exposure to trastuzumab. The finding that both P-Tyr levels and HER2 levels in each of these assays reveal no change further supports the conclusions derived from the gene mRNA expression array data [33]. Thus, examination of HER2 steady-state protein levels and key phosphorylation sites [10] did not reveal the molecular perturbation responsible for well-documented trastuzumab inhibition of cell proliferation and downstream pathways.

In contrast to trastuzumab, lapatinib treatment almost completely abrogates the HER2-P-Tyr as a signalling element while leaving HER2 protein levels unchanged. Thus, lapatinib acts through the blockage of tyrosine phosphorylation rather than the modulation of internalization or metabolism of the HER2 protein. We further show a sequential relationship between lapatinib-induced blockage of HER2-P-Tyr (8 and 48 hours) and induction of cell death at day 5-6 of drug exposure. At this late time point, MTT, Tritiated thymidine, actin levels and microscopy all support a lapatinib induction of cell lyses.

The inhibition of cell growth was used to define the conditions of ongoing drug-induced biological effects. The cell growth experiments described here are in agreement with similar reports by others [24,31]. Trastuzumab induced slower growth but did not suppress net increases in cell numbers over time. This *in vitro* effect of trastuzumab parallels the clinical observation that a prolonged stable disease is a common outcome of treatment [37].

Treatment of amplicon-positive breast cancer cells with cetuximab confirms reports by others that anti-EGFR therapy does not inhibit proliferation, induce cell death, nor inhibit HER2 phosphorylation in these cell lines [31]. Furthermore, we did not find that combinations of cetuximab and trastuzumab augment inhibition of cell growth or promote apoptotic events [31]. Cell growth inhibition and phosphorylation experiments with cetuximab have not been in agreement with results of similar investigations using small molecules with EGFR tyrosine kinase inhibitory activity (TKI) [38,39]. However, drugs such as gefitinib, which are believed to be relatively specific for EGFR, are also found to block HER2 phosphorylation at higher doses [38], complicating the interpretation of its mechanism of action [38]. Whether there is an independent anti-EGFR activity crossinhibiting HER2 phosphorylation (cross-talk) or whether this observation is a consequence of the direct anti-HER2-phosphorylation activity of gefitinib is unclear. Results of these *in*  *vitro* experiments may mislead investigators into attributing findings to cross-talk between receptor pathways rather than cross-reactivity of the reagent tested. We may over estimate biologic activity when reagents are used at too high of a concentration or too long of an exposure. Results of these experiments may mislead investigators into attributing findings to cross-talk between receptor pathways rather than the cross reactivity of the reagent tested. Thus, we conclude that results of experiments testing cetuximab's biologic effects more closely represent, and are more specific to, the pure anti-EGFR blockade. We clearly show here, in agreement with previous reports by others that pure anti-EGFR1 inhibition of signalling does not result in cell death or modulation of HER2 phosphorylation levels [29].

366 Protein Phosphorylation in Human Health

proliferation and downstream pathways.

membrane HER2 levels could not be implicated as a mechanism to down-regulate HER2 signalling. Despite these observations for HER2, our gene mRNA expression array data did suggest numerous intracellular pathway modulations mediated by trastuzumab binding to the target cells examined here [33]. These findings confirm transmembrane signal transduction modulation directed and specific to trastuzumab binding [33-35]. For example, we found that, the ubiquitin pathway is up-regulated upon trastuzumab binding to HER2

Trastuzumab has not been definitively shown to retain the tyrosine kinase inhibitory activity of the parent 4D5 mouse monoclonal antibody [23]. Here we show that the level of HER2-P-Tyr/Thr corrected to cell number, actin levels, and the steady-state levels of intact extractable HER2, is not altered by exposure to trastuzumab. The finding that both P-Tyr levels and HER2 levels in each of these assays reveal no change further supports the conclusions derived from the gene mRNA expression array data [33]. Thus, examination of HER2 steady-state protein levels and key phosphorylation sites [10] did not reveal the molecular perturbation responsible for well-documented trastuzumab inhibition of cell

In contrast to trastuzumab, lapatinib treatment almost completely abrogates the HER2-P-Tyr as a signalling element while leaving HER2 protein levels unchanged. Thus, lapatinib acts through the blockage of tyrosine phosphorylation rather than the modulation of internalization or metabolism of the HER2 protein. We further show a sequential relationship between lapatinib-induced blockage of HER2-P-Tyr (8 and 48 hours) and induction of cell death at day 5-6 of drug exposure. At this late time point, MTT, Tritiated

The inhibition of cell growth was used to define the conditions of ongoing drug-induced biological effects. The cell growth experiments described here are in agreement with similar reports by others [24,31]. Trastuzumab induced slower growth but did not suppress net increases in cell numbers over time. This *in vitro* effect of trastuzumab parallels the clinical

Treatment of amplicon-positive breast cancer cells with cetuximab confirms reports by others that anti-EGFR therapy does not inhibit proliferation, induce cell death, nor inhibit HER2 phosphorylation in these cell lines [31]. Furthermore, we did not find that combinations of cetuximab and trastuzumab augment inhibition of cell growth or promote apoptotic events [31]. Cell growth inhibition and phosphorylation experiments with cetuximab have not been in agreement with results of similar investigations using small molecules with EGFR tyrosine kinase inhibitory activity (TKI) [38,39]. However, drugs such as gefitinib, which are believed to be relatively specific for EGFR, are also found to block HER2 phosphorylation at higher doses [38], complicating the interpretation of its mechanism of action [38]. Whether there is an independent anti-EGFR activity crossinhibiting HER2 phosphorylation (cross-talk) or whether this observation is a consequence of the direct anti-HER2-phosphorylation activity of gefitinib is unclear. Results of these *in*

thymidine, actin levels and microscopy all support a lapatinib induction of cell lyses.

observation that a prolonged stable disease is a common outcome of treatment [37].

[35], as has been reported by others through ligase activity at tyrosine 1112 [35,36].

These observations suggest that the anti-EGFR1 tyrosine kinase effects of lapatinib are not contributing to its anti-HER2 effects. Thus, the results of the panel of anti-HER2-P-Tyrantibodies demonstrating almost complete abrogation of HER2 phosphorylation by lapatinib and its associated delayed cell cytotoxicity (day 5-6) establishes a specific and unique link between HER2 tyrosine phosphorylation and cell survival. Importantly, these results also suggest that additional signalling through HER3 and HER4 may not be critical for apoptosis [40], except as it may ultimately be mediated through HER2 tyrosine kinase inhibition (reduction in hetero-dimerization) [40]. Thus, blocking dimerization by itself, which is unlikely to affect steric change in the intracellular domain, will not induce a modulation of P-Tyr levels. Taken together, these data suggest that testing the effect of a specific and robust HER2 tyrosine kinase inhibitory reagent is warranted.

In the clinic, where the anti-EGFR toxicity of lapatinib is dose limiting (diarrhea and skin rash) and prohibits dose escalation, the testing of a small molecule TKI manifesting pure HER2 kinase inhibition would be of great interest. Recently a pan ErbB TKI was tested and shown to be dose limited by the EGFR-mediated bowel toxicity, further supporting the conclusions presented here [41]. A pure anti-HER2 agent would be even more compelling if one could demonstrate upon dose escalation increased tumor cytotoxicity and a lack of other intervening non-HER2 related dose-limiting toxicities. Close examination of many of the TKIs demonstrate widespread promiscuity with many unexpected cross-reactivities [42]. In general, clinical success of a widely reactive kinase inhibitor is not informative as an identifier of the key pathway(s) responsible for mediating anti-tumor effects and often leads to "assumed" mechanisms of activity.

Using the four anti-P-Tyr sera, we could not find any diminution in the phosphorylation levels as assayed by Western blots, in cells treated up to nine days in the presence of trastuzumab. In addition, the anti-phospho-threonine reagent was used as a negative control, and was found not to be blocked by lapatinib demonstrating the specificity of the drug and the kinase activity. Thus, we propose that the kinase inhibition that was originally described by the mouse antibody 4D5 [23] has been lost in the humanization process [25]. The abundance of well-documented evidence that inhibition of specific downstream pathways is a consequence of trastuzumab-cell binding remains unexplained.

Modulation of HER2 Tyrosine/Threonine Phosphorylation and Cell Signalling 369

One hypothesis to be considered is that the trastuzumab effect is a weaker version of what is seen with lapatinib and while we cannot demonstrate a small loss of P-Tyr activity in culture, more sensitive techniques might be informative. Alternatively, trastuzumab inhibition may be allosteric or conformational in nature and at the level of the cytoplasmic signal transducers [10] ability to detect the phosphate moieties in the context of an altered peptide conformation [43]. One may further speculate that due to the unique binding epitope of trastuzumab, which is in extra-cellular proximal domain, trans-membrane tertiary structural changes may be induced. Cross-linking HER2 (dimers) at the extracellular proximal domain may disrupt the P-Tyr access to cytoplasmic signal transducers by physical constraints. This hypothesis would suggest that other membrane receptor systems

Our results demonstrate the presence of a small 20 kD protein concentrated in the extracted membrane fraction which is immunologically similar to an extra-cellular segment of HER2 protein. This may provide further evidence of specific enzymatic digestion of HER2 into novel biologic agents [44,45]. The low molecular weight of this identified fragment and its association with the membrane fraction raises the possibility that it may function as a ligand for another, as yet unidentified, receptor. Thus, we hypothesize that HER2 may not be a classical receptor as has been suspected by the lack of a ligand binding moiety but rather a pre-ligand entity which requires regulated and specific digestion to be activated. The differential expression of this moiety with lapatinib in contrast to trastuzumab treatment, demonstrates the complexity of this system and the difficulty in attributing a single

These observations are not informative regarding the precise cytotoxic mechanism of trastuzumab activity in the clinic. Models based on preclinical animal studies favor immune mechanisms as key in mediating tumor shrinkage [46]. Studies of patients treated in the neoadjuvant setting also support these findings [43]. However the clinical effects of trastuzumab could be divided into two distinct effects, a growth inhibitory effect as seen in vitro and which manifests clinically as long term stable disease and a cytotoxic effect as one might expect from immune mediated cell lysis which results in rapid tumor shrinkage in the

In addition to these hypotheses, there is evidence that HER2 induced downstream effects actually amplify pro-inflammatory factors such as COX-2 [22] creating a micro-environment for promoting immune cell mediated tumor cell killing. These data are in agreement with modulation of a prostaglandin pathway identified by gene mRNA expression array experiments [33] in trastuzumab treated cells. Thus downstream events induced by HER2 or regulated by trastuzumab binding, may promote the immune mediated killing of tumor cells by trastuzumab. These findings provide a strong rational for combining other antibreast cancer cell antibodies with trastuzumab therapy. Furthermore the additional antitumor efficacy observed with combinations of trastuzumab and chemotherapy could also be attributable to the known effects of certain chemotherapeutic agents, which render tumor

could also be modulated in a similar fashion.

molecular mechanism in transmembrane signal transduction.

clinic, especially as seen in neo-adjuvant studies [47].

**Figure 2.** Demonstrates a series of experiments evaluating a 20 kD protein detected by anti-HER2 extracellular domain (AA 182-373) anti-sera at 48 hours. *Lanes 1-5* demonstrate the full Western blot gel of BT-474 (48 hours, from Figure 1) showing two bands: the p185 HER2 protein and the 20 kD protein (arrow on left) *lanes 1-4*. Note that this protein in not detected in *lane 5* which represents lapatinibtreated cells, while the p185 HER2 is mildly reduced in *lane 5*. *Lanes 1- 14* show the corresponding actin control confirming that equal numbers of cell were applied to *lanes 1-5*. Exposing BT-475to cells to an acid wash of pH 4.0 does not elute the protein as *lane 7* is the acid wash and the cell pellet that remains is shown in *lane 6*, has both p185 and the 20 kD protein. Using the Triton X Perfect Focus Membrane Protein kit methodology shows a clearer picture of localization of the 20 kD band with membrane HER2 protein and the 20 kD band (arrow) both in *lane 8* (membrane protein concentrate) and corresponding cell pellet, run in *lane 9*, is negative.

One hypothesis to be considered is that the trastuzumab effect is a weaker version of what is seen with lapatinib and while we cannot demonstrate a small loss of P-Tyr activity in culture, more sensitive techniques might be informative. Alternatively, trastuzumab inhibition may be allosteric or conformational in nature and at the level of the cytoplasmic signal transducers [10] ability to detect the phosphate moieties in the context of an altered peptide conformation [43]. One may further speculate that due to the unique binding epitope of trastuzumab, which is in extra-cellular proximal domain, trans-membrane tertiary structural changes may be induced. Cross-linking HER2 (dimers) at the extracellular proximal domain may disrupt the P-Tyr access to cytoplasmic signal transducers by physical constraints. This hypothesis would suggest that other membrane receptor systems could also be modulated in a similar fashion.

368 Protein Phosphorylation in Human Health

cell pellet, run in *lane 9*, is negative.

**Figure 2.** Demonstrates a series of experiments evaluating a 20 kD protein detected by anti-HER2 extracellular domain (AA 182-373) anti-sera at 48 hours. *Lanes 1-5* demonstrate the full Western blot gel of BT-474 (48 hours, from Figure 1) showing two bands: the p185 HER2 protein and the 20 kD protein (arrow on left) *lanes 1-4*. Note that this protein in not detected in *lane 5* which represents lapatinibtreated cells, while the p185 HER2 is mildly reduced in *lane 5*. *Lanes 1- 14* show the corresponding actin control confirming that equal numbers of cell were applied to *lanes 1-5*. Exposing BT-475to cells to an acid wash of pH 4.0 does not elute the protein as *lane 7* is the acid wash and the cell pellet that remains is shown in *lane 6*, has both p185 and the 20 kD protein. Using the Triton X Perfect Focus Membrane Protein kit methodology shows a clearer picture of localization of the 20 kD band with membrane HER2 protein and the 20 kD band (arrow) both in *lane 8* (membrane protein concentrate) and corresponding

Our results demonstrate the presence of a small 20 kD protein concentrated in the extracted membrane fraction which is immunologically similar to an extra-cellular segment of HER2 protein. This may provide further evidence of specific enzymatic digestion of HER2 into novel biologic agents [44,45]. The low molecular weight of this identified fragment and its association with the membrane fraction raises the possibility that it may function as a ligand for another, as yet unidentified, receptor. Thus, we hypothesize that HER2 may not be a classical receptor as has been suspected by the lack of a ligand binding moiety but rather a pre-ligand entity which requires regulated and specific digestion to be activated. The differential expression of this moiety with lapatinib in contrast to trastuzumab treatment, demonstrates the complexity of this system and the difficulty in attributing a single molecular mechanism in transmembrane signal transduction.

These observations are not informative regarding the precise cytotoxic mechanism of trastuzumab activity in the clinic. Models based on preclinical animal studies favor immune mechanisms as key in mediating tumor shrinkage [46]. Studies of patients treated in the neoadjuvant setting also support these findings [43]. However the clinical effects of trastuzumab could be divided into two distinct effects, a growth inhibitory effect as seen in vitro and which manifests clinically as long term stable disease and a cytotoxic effect as one might expect from immune mediated cell lysis which results in rapid tumor shrinkage in the clinic, especially as seen in neo-adjuvant studies [47].

In addition to these hypotheses, there is evidence that HER2 induced downstream effects actually amplify pro-inflammatory factors such as COX-2 [22] creating a micro-environment for promoting immune cell mediated tumor cell killing. These data are in agreement with modulation of a prostaglandin pathway identified by gene mRNA expression array experiments [33] in trastuzumab treated cells. Thus downstream events induced by HER2 or regulated by trastuzumab binding, may promote the immune mediated killing of tumor cells by trastuzumab. These findings provide a strong rational for combining other antibreast cancer cell antibodies with trastuzumab therapy. Furthermore the additional antitumor efficacy observed with combinations of trastuzumab and chemotherapy could also be attributable to the known effects of certain chemotherapeutic agents, which render tumor

cells more sensitive to immune attack [48]. If this is the case, research should be focused on mechanisms by which chemotherapy may damage tumor cells sufficiently to allow more efficient killing by immune mechanisms rather than focus on apoptotic pathways that enhance chemotherapy cytotoxic effects.

Modulation of HER2 Tyrosine/Threonine Phosphorylation and Cell Signalling 371

[6] Desmedt, C Sperindle, J Piette, F Huang, W Jin, X. *et al*. Quantitation of HER2 expression or HER2:HER2 dimers and differential survival in a cohort of metastatic breast cancer patients carefully for trastuzumab treatment primarily by FISH. *Diagn Mol Pathol*. 18:22-

[7] Wolf-Yardin, A Kumer, N Zhang, Y Hautaniememi, S Zaman, M *et al*. Effects of HER2 overexpression on cell signalling networks governing proliferation and migration. *Mol*

[8] Yuan, CX Lasut, AL Wynn, R Neff, NT Hollis, GF. Purification of Her-2 extracellular domain and identification of its cleavage site. *Protein Expr Purif*.29:217-22, 2003 [9] Molina, MA Codony-Servat, J Albanell, J Rojo, F Arribas, J. *et al*. Trastuzumab (herceptin), a humanized anti-Her2 receptor monoclonal antibody, inhibits basal and activated Her2 ectodomain cleavage in breast cancer cells. *Cancer Res*. 61: 4744-4749,

[10] Dankort D, Jeyabalan N, Jones N, Dumont DJ, Muller WJ. Multiple ErbB-2/Neu Phosphorylation Sites Mediate Transformation through Distinct Effector Proteins. J Biol

[11] Zhou, BP Liao, Y Xia, W Spohn, L Lee, MH. *et al*. Cytoplasmic localization of p21 Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-over expressing cells.

[12] Fedi, P Pierce, JH di Fiore, PP. Efficient coupling with phosphotidylinositol 3-kinase, but not phospholipase C gamma or GTPase-activating protein, distinguishes ERB-3 signalling from that of other ERB/EGFR family receptors. *Mol Cell Biol* 14:492-500, 1994. [13] Holbro T, Beerli RR, Maurer F, et al The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast cell proliferation. Proc Natl Acad

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## **Author details**

Yamuna D. Gandaharen and Sydney Welt *Welt Bio-Molecular Pharmaceutical, LLC. Molecular Research, Armonk, NY, U.S.A.*

Rachel S. Welt *Department of Biological Sciences, Fordham University, NYC, NY, U.S.A.*

David Kostyal *ARD Laboratories, Biologic Research, Akron, OH, U.S.A.*

## **Acknowledgement**

Funding and support-PA State Department of Health Project Cure Award SAP #<PHONE_NUMBER> and the Cantwell Foundation Fund of Coudersport Pennsylvania 16901.

## **Disclosure statement**

The authors declare that there are no competing financial interests in carrying out this study.

## **5. References**

[6] Desmedt, C Sperindle, J Piette, F Huang, W Jin, X. *et al*. Quantitation of HER2 expression or HER2:HER2 dimers and differential survival in a cohort of metastatic breast cancer patients carefully for trastuzumab treatment primarily by FISH. *Diagn Mol Pathol*. 18:22- 29, 2009.

370 Protein Phosphorylation in Human Health

**Author details**

Rachel S. Welt

David Kostyal

study.

**5. References**

1997.

2:127-137, 2001.

**Acknowledgement**

**Disclosure statement**

enhance chemotherapy cytotoxic effects.

Yamuna D. Gandaharen and Sydney Welt

*ARD Laboratories, Biologic Research, Akron, OH, U.S.A.*

receptor interactions. *Embo J* 15: 2452-2467, 1996.

*Acad Sci U S A* 95:6809-6814, 1998.

*J Biol Chem* 274:8865-8874, 1999.

cells more sensitive to immune attack [48]. If this is the case, research should be focused on mechanisms by which chemotherapy may damage tumor cells sufficiently to allow more efficient killing by immune mechanisms rather than focus on apoptotic pathways that

Funding and support-PA State Department of Health Project Cure Award SAP #<PHONE_NUMBER>

The authors declare that there are no competing financial interests in carrying out this

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*Welt Bio-Molecular Pharmaceutical, LLC. Molecular Research, Armonk, NY, U.S.A.*

*Department of Biological Sciences, Fordham University, NYC, NY, U.S.A.*

and the Cantwell Foundation Fund of Coudersport Pennsylvania 16901.

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**Section 4**

**Protein Kinases and Phosphatases**

**in Cell Cycle Regulation**

**Protein Kinases and Phosphatases in Cell Cycle Regulation**

374 Protein Phosphorylation in Human Health

[48] Segerling M, Ohanian SH, Boros T. Chemotherapeutic drugs increase killing of tumor

cells by antibody and complement. Science 188: 55-57, 1975.

**Chapter 13**

© 2012 Fraschini et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Protein Phosphorylation is an Important Tool**

Protein phosphorylation is a reversible posttranslational modification that can modulate protein role in several physiological processes in almost every possible way. These include modification of its intrinsic biological activity, subcellular location, half-life and binding with other proteins. Protein phosphorylation is particularly important for the regulation of

Protein phosphorylation is the covalent binding of a phosphate group to some critical residues of the polypeptide. The phosphorylation state of a protein is given by a balance between the activity of protein kinases and protein phosphatases. Eukaryotic protein kinases transfer phosphate groups (PO43-) from ATP to an hydroxyl group of the lateral chain of specific serine, threonine or tyrosine residues on peptide substrates. In simple eukaryotic cells, like yeasts, Ser/Thr kinases are more common, while more complex eukaryotic cells, like human cells, have many Tyr kinases. Protein kinases recognize their substrates specifically and their active site consists of an activation loop and a catalytic loop between which substrates bind. Protein kinases differ from each other in the structure of their catalitic domain. A second class of enzymes, protein phosphatases, is responsible for the reverse reaction in which phosphate groups are removed from a protein. Phosphatases gain specificity by binding protein cofactors which facilitate binding to specific phosphoproteins. The active phosphatase often consists of a

The use of protein phosphorylation/dephosphorylation as a control mechanism has many advantages since it is rapid, it does not require new proteins to be made or degraded and it

and reproduction in any medium, provided the original work is properly cited.

**to Change the Fate of Key Players in**

**the Control of Cell Cycle Progression**

Roberta Fraschini, Erica Raspelli and Corinne Cassani

key proteins involved in the control of cell cycle progression.

complex of the phosphatase catalytic subunit and a regulatory subunit.

**in** *Saccharomyces cerevisiae*

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/47809

**1. Introduction**

## **Protein Phosphorylation is an Important Tool to Change the Fate of Key Players in the Control of Cell Cycle Progression in** *Saccharomyces cerevisiae*

Roberta Fraschini, Erica Raspelli and Corinne Cassani

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/47809

## **1. Introduction**

Protein phosphorylation is a reversible posttranslational modification that can modulate protein role in several physiological processes in almost every possible way. These include modification of its intrinsic biological activity, subcellular location, half-life and binding with other proteins. Protein phosphorylation is particularly important for the regulation of key proteins involved in the control of cell cycle progression.

Protein phosphorylation is the covalent binding of a phosphate group to some critical residues of the polypeptide. The phosphorylation state of a protein is given by a balance between the activity of protein kinases and protein phosphatases. Eukaryotic protein kinases transfer phosphate groups (PO43-) from ATP to an hydroxyl group of the lateral chain of specific serine, threonine or tyrosine residues on peptide substrates. In simple eukaryotic cells, like yeasts, Ser/Thr kinases are more common, while more complex eukaryotic cells, like human cells, have many Tyr kinases. Protein kinases recognize their substrates specifically and their active site consists of an activation loop and a catalytic loop between which substrates bind. Protein kinases differ from each other in the structure of their catalitic domain. A second class of enzymes, protein phosphatases, is responsible for the reverse reaction in which phosphate groups are removed from a protein. Phosphatases gain specificity by binding protein cofactors which facilitate binding to specific phosphoproteins. The active phosphatase often consists of a complex of the phosphatase catalytic subunit and a regulatory subunit.

The use of protein phosphorylation/dephosphorylation as a control mechanism has many advantages since it is rapid, it does not require new proteins to be made or degraded and it

© 2012 Fraschini et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

is easily reversible. The phosphorylation of specific residues induces structural changes that regulate protein functions by modulating protein folding, substrate affinity, stability and activity. For example, phosphorylation can cause switch-like changes in protein function, which can also lead to major modifications in the catalytic function of enzymes, including kinases and phosphatases. In addition, protein phosphorylation often leads to rearrangement in the structure of the protein that can induce changes in interacting partners or subcellular localization.

Protein Phosphorylation is an Important Tool to Change the Fate

of Key Players in the Control of Cell Cycle Progression in *Saccharomyces cerevisiae* 379

activates its catalitic domain and allows its binding to specific substrates. Polo-like kinases have a conserved Ser/Thr kinase domain and a noncatalytic C-terminal region composed by two homologous boxes Polo Box Domain (PBD) [3], that appear to target the kinase to mitotic substrates. In addition, it has been shown that phosphopeptide binding to the PBD stimulates kinase activity, suggesting a conformational switching mechanism for Plk

It is the simplicity, reversibility and flexibility of phosphorylation that explains why it has been adopted as the most general control mechanism of the cell. Below we describe how phosphorylation and dephosphorylation events can finely regulate in space and time some

Budding yeast cyclin-dependent kinase Cdk1 is the motor that drives cell cycle progression. Cdk1 activity is regulated at multiple levels in different cell cycle phases by interactions with different proteins, called cyclins, and by phosphorylation and dephosphorylation events. In G2 phase of the cell cycle, the protein kinase Swe1 inhibits Cdk1 by phosphorylating the conserved Y19 residue of its catalytic subunit Cdc28 [4], thus blocking both switch from polar to isotropic bud growth and nuclear division, since both these events rely on G2/M Cdk activity. This inhibitory phosphorylation is reversed by the Mih1 phosphatase [5], leading to Cdk1 activation and entry into mitosis. Swe1 phosphorylates and inhibits Cdc28 during every cell cycle and in case of problems in the bud neck, bud formation, actin cytoskeleton and abnormal cell shape. Successful bud formation leads to Swe1 degradation in late G2 phase, while morphological defects block this degradation, thus delaying entry into mitosis. Swe1 has therefore a critical role in coordinating cell

Swe1 levels are controlled by the "morphogenesis checkpoint", a pathway that is activated in response to alterations in the actin cytoskeleton or in septin organization. The activation of this checkpoint ultimately leads to Swe1 stabilization and a subsequent delay in nuclear division [6]. In an unperturbed cell cycle, Swe1 is recruited to the mother-bud neck in S phase; this change in its localization, which is promoted by the interaction with its regulators Hsl1 and Hsl7, is essential for subsequent Swe1 multiphosphorylation, an event that leads to its ubiquitylation and degradation, thus allowing entry into mitosis. The morphogenesis checkpoint causes Swe1 stabilization by interfering with its localization to the bud neck, acting directly on Swe1 or on its regulators Hsl1 and Hsl7, thus preventing modifications that lead to its degradation [7]. Accordingly, the lack of septin localization at the bud neck results in Swe1 stabilization [7-9], and even subtle perturbations in septin structure interfere with Hsl1 and Swe1 localization to the bud neck [7]. Moreover, actin depolymerization in budded cells causes both stabilization of Swe1 and its displacement from the bud neck without altering Hsl1 localization [7], indicating that morphogenesis checkpoint activation prevents Swe1 degradation by interfering with Swe1 localization to

key proteins in the control of cell cycle progression in *Saccharomyces cerevisiae* .

**2. The protein kinase Swe1 is regulated at several levels**

regulation and a double function for the PBD.

morphogenesis with nuclear division.

the bud neck, thus inhibiting its phosphorylation.

Phosphorylation acts as a molecular switch for many regulatory events in signalling pathways that drive cell division, proliferation, differentiation and apoptosis. In order to ensure an appropriate balance of protein phosphorylation, the cell can compartmentalize both protein kinases and phosphatases. Another kind of regulation can be achieved by the spatial distribution of kinases and phosphatases, that creates a gradient of phosphorylated substrates across different subcellular compartments. This spatial separation can also control the activity of other proteins or enzymes and the occurrence of other posttranslational modifications.

Even in a simple organism like budding yeast, approximately 3% of its proteins are kinases or phosphatases. Some of these enzymes are extremely specific, indeed that are able to phosphorylate or dephosphorylate only a few target proteins, while others can act broadly on many proteins. The examples of known targets of phosphorylation include most protein components of the cell like enzymes, structural proteins, cell receptors, ion channels and signaling factors. If a protein is controlled by its phosphorylation state, its activity will be directly dependent on the activity of the kinases and phosphatases that act on it. It is quite common for a phosphate group to be added or removed from a protein continually, a cycle that allows a protein to switch rapidly from one state to another.

A protein can be modified by the addition of a single phosphate group or it can undergo multisite phosphorylation, these events can be driven by a single kinase or by multiple kinases that act in concert. Multisite phosphorylation can determine the extent and duration of a cellular response and can integrate multiple signals on the same protein. For example, many protein kinases involved in the cell cycle control function by generating phosphoSer/Thr-containing sequence motifs in their substrates that are then recognized by phosphoSer/Thr binding proteins. In several cases the phosphopeptide binding domain targets the kinase to prephosphorylated (primed) sites and then mediates processive phosphorylation of the substrate. An important example of this regulation is given by some budding yeast proteins that are phosphorylated by the catalitic subunit of the Cyclin-Dependent Kinase (CDK), Cdc28, that "primes" the protein in order to bind and to be phosphorylated by the Polo-like kinase (Plk) Cdc5.

Cdks are the master regulators of the cell cycle and Cdc5 plays key roles during all stages of mitosis and in the cell cycle checkpoint response to genotoxic stress [1,2]. The protein kinase Cdc28 drives every cell cycle transition and its activity is tightly regulated by phosphorylation/dephosphorylation events and by its association with a class of proteins called cyclins. Cyclins levels fluctuate during the cell cycle and their binding to Cdc28 activates its catalitic domain and allows its binding to specific substrates. Polo-like kinases have a conserved Ser/Thr kinase domain and a noncatalytic C-terminal region composed by two homologous boxes Polo Box Domain (PBD) [3], that appear to target the kinase to mitotic substrates. In addition, it has been shown that phosphopeptide binding to the PBD stimulates kinase activity, suggesting a conformational switching mechanism for Plk regulation and a double function for the PBD.

It is the simplicity, reversibility and flexibility of phosphorylation that explains why it has been adopted as the most general control mechanism of the cell. Below we describe how phosphorylation and dephosphorylation events can finely regulate in space and time some key proteins in the control of cell cycle progression in *Saccharomyces cerevisiae* .

## **2. The protein kinase Swe1 is regulated at several levels**

378 Protein Phosphorylation in Human Health

or subcellular localization.

modifications.

is easily reversible. The phosphorylation of specific residues induces structural changes that regulate protein functions by modulating protein folding, substrate affinity, stability and activity. For example, phosphorylation can cause switch-like changes in protein function, which can also lead to major modifications in the catalytic function of enzymes, including kinases and phosphatases. In addition, protein phosphorylation often leads to rearrangement in the structure of the protein that can induce changes in interacting partners

Phosphorylation acts as a molecular switch for many regulatory events in signalling pathways that drive cell division, proliferation, differentiation and apoptosis. In order to ensure an appropriate balance of protein phosphorylation, the cell can compartmentalize both protein kinases and phosphatases. Another kind of regulation can be achieved by the spatial distribution of kinases and phosphatases, that creates a gradient of phosphorylated substrates across different subcellular compartments. This spatial separation can also control the activity of other proteins or enzymes and the occurrence of other posttranslational

Even in a simple organism like budding yeast, approximately 3% of its proteins are kinases or phosphatases. Some of these enzymes are extremely specific, indeed that are able to phosphorylate or dephosphorylate only a few target proteins, while others can act broadly on many proteins. The examples of known targets of phosphorylation include most protein components of the cell like enzymes, structural proteins, cell receptors, ion channels and signaling factors. If a protein is controlled by its phosphorylation state, its activity will be directly dependent on the activity of the kinases and phosphatases that act on it. It is quite common for a phosphate group to be added or removed from a protein continually, a cycle

A protein can be modified by the addition of a single phosphate group or it can undergo multisite phosphorylation, these events can be driven by a single kinase or by multiple kinases that act in concert. Multisite phosphorylation can determine the extent and duration of a cellular response and can integrate multiple signals on the same protein. For example, many protein kinases involved in the cell cycle control function by generating phosphoSer/Thr-containing sequence motifs in their substrates that are then recognized by phosphoSer/Thr binding proteins. In several cases the phosphopeptide binding domain targets the kinase to prephosphorylated (primed) sites and then mediates processive phosphorylation of the substrate. An important example of this regulation is given by some budding yeast proteins that are phosphorylated by the catalitic subunit of the Cyclin-Dependent Kinase (CDK), Cdc28, that "primes" the protein in order to bind and to be

Cdks are the master regulators of the cell cycle and Cdc5 plays key roles during all stages of mitosis and in the cell cycle checkpoint response to genotoxic stress [1,2]. The protein kinase Cdc28 drives every cell cycle transition and its activity is tightly regulated by phosphorylation/dephosphorylation events and by its association with a class of proteins called cyclins. Cyclins levels fluctuate during the cell cycle and their binding to Cdc28

that allows a protein to switch rapidly from one state to another.

phosphorylated by the Polo-like kinase (Plk) Cdc5.

Budding yeast cyclin-dependent kinase Cdk1 is the motor that drives cell cycle progression. Cdk1 activity is regulated at multiple levels in different cell cycle phases by interactions with different proteins, called cyclins, and by phosphorylation and dephosphorylation events. In G2 phase of the cell cycle, the protein kinase Swe1 inhibits Cdk1 by phosphorylating the conserved Y19 residue of its catalytic subunit Cdc28 [4], thus blocking both switch from polar to isotropic bud growth and nuclear division, since both these events rely on G2/M Cdk activity. This inhibitory phosphorylation is reversed by the Mih1 phosphatase [5], leading to Cdk1 activation and entry into mitosis. Swe1 phosphorylates and inhibits Cdc28 during every cell cycle and in case of problems in the bud neck, bud formation, actin cytoskeleton and abnormal cell shape. Successful bud formation leads to Swe1 degradation in late G2 phase, while morphological defects block this degradation, thus delaying entry into mitosis. Swe1 has therefore a critical role in coordinating cell morphogenesis with nuclear division.

Swe1 levels are controlled by the "morphogenesis checkpoint", a pathway that is activated in response to alterations in the actin cytoskeleton or in septin organization. The activation of this checkpoint ultimately leads to Swe1 stabilization and a subsequent delay in nuclear division [6]. In an unperturbed cell cycle, Swe1 is recruited to the mother-bud neck in S phase; this change in its localization, which is promoted by the interaction with its regulators Hsl1 and Hsl7, is essential for subsequent Swe1 multiphosphorylation, an event that leads to its ubiquitylation and degradation, thus allowing entry into mitosis. The morphogenesis checkpoint causes Swe1 stabilization by interfering with its localization to the bud neck, acting directly on Swe1 or on its regulators Hsl1 and Hsl7, thus preventing modifications that lead to its degradation [7]. Accordingly, the lack of septin localization at the bud neck results in Swe1 stabilization [7-9], and even subtle perturbations in septin structure interfere with Hsl1 and Swe1 localization to the bud neck [7]. Moreover, actin depolymerization in budded cells causes both stabilization of Swe1 and its displacement from the bud neck without altering Hsl1 localization [7], indicating that morphogenesis checkpoint activation prevents Swe1 degradation by interfering with Swe1 localization to the bud neck, thus inhibiting its phosphorylation.

Swe1 is subjected to multiple regulations that change its phosphorylation state (35-40 phosphorylated sites have been identified *in vivo*), subcellular localization and protein levels (Figure 1). During S phase, Swe1 accumulates in the nucleus where it is phosphorylated by Clb-Cdc28 before being exported to the cytoplams and then to the daughter side of the bud neck. Swe1 nuclear export is essential for its degradation in G2/M in fact a Swe1 variant that cannot be exported from the nucleus is largely stabilized [10]. Clb-Cdc28 phosphorylates Swe1 at multiple sites [11] and these events can occur in the nucleus, in the cytoplasm or at the bud neck. This multiphosphorylation by Cdc28-Clb has different roles: on one hand it promotes Swe1 activity generating a feedback loop, while on the other hand it promotes Swe1 degradation [12]. At the mother-bud neck, filaments of conserved proteins called septins (Cdc3, Cdc10, Cdc11, Cdc12 and Shs1 in *S. cerevisiae*) form a dynamic ring structure [13] that is essential for the recruitment of a number of proteins involved in the control of cell cycle progression [14,15]. Among these, the septin ring acts as a platform to recruit several Swe1 regulators, such as the Hsl1 protein kinase and its adaptor Hsl7, both essential for Swe1 localization to the bud neck and for its phosphorylation [7,9]. Hsl1, whose kinase activity requires assembled septins, undergoes autophosphorylation [8,16] and phosphorylates Hsl7 [9] but does not seem to phosphorylates Swe1 [17] although its kinase activity is required for Swe1 recruitment at the bud neck [18]. The fact that Hsl1 activation requires assembled septins ensures that Swe1 degradation does not begin until a bud has formed, thus providing a link between bud formation and entry into mitosis [18]. Also the PAK (p21-activated kinase) kinase Cla4 associates with the septin ring and is involved in Swe1 phosphorylation, probably during S phase [19]. Moreover Swe1 phosphorylation by Clb-Cdc28 promotes subsequent phosphorylation by the Polo-like kinase Cdc5; in fact the synergistic phosphorylation that can be observed *in vitro* on Swe1 by Clb2-Cdc28 and Cdc5 is the result of priming Swe1 by Cdc28, in which the resulting phosphorylated Swe1 becomes a better substrate for Cdc5 [20]. An additional level of Swe1 regulation involves the Cdc55 regulatory subunit of protein phosphatase PP2A, that has been implicated in its degradation since loss of Cdc55 function causes Swe1 stabilization [21]. However, how this control happens at the molecular level is not clear. In addition, a mathematical model for the morphogenesis checkpoint activation suggested that a subset of Swe1 phosphorylations could inhibit its activity whereas other phosphorylations could target Swe1 for degradation [22]. In any case, hyperphosphorylated Swe1 species are recognized by a still unidentified ubiquitin ligase and ubiquitylated [23]. Subsequently, Swe1 is degraded via the proteasome and this event allows mitotic entry [23]. However, how bud neck-localized Swe1 is targeted to degradation after phosphorylation is still obscure. There are Swe1 variants that do not undergo degradation although they show proper bud neck localization, phosphorylation and interaction with known Swe1 regulators [23], indicating that still unknown Swe1 regulators exist.

Protein Phosphorylation is an Important Tool to Change the Fate

of Key Players in the Control of Cell Cycle Progression in *Saccharomyces cerevisiae* 381

frequently found in DNA repair and checkpoint proteins [25,26] and RING domains are typical of E3 ubiquitin ligases [26]. The presence of an FHA domain implies that one or more protein kinases function upstream of these proteins. An intact FHA domain is required for checkpoint function of all characterized FHA-RING ubiquitin ligases. All these proteins appear to control different aspects of the mitotic cell cycle, but several molecular details of their functions are still obscure [for review, see 27]. In particular, the *S. cerevisiae* Dma proteins are involved in mitotic checkpoints and contribute to control septin ring dynamics and cytokinesis by unknown mechanisms [28,29]. Moreover, *in vitro* ubiquitin ligase activity of Dma1 and Dma2 has been described [30], although their *in vivo* targets are still unknown.

**Figure 1.** Model for Swe1 regulation. Swe1 shuttles from the nucleus to the cytoplasm and then it is translocated to the septin ring at the bud neck. These events are controlled through phosphorylation by Cdc28-Clb kinase. Once at the bud neck, Swe1 undergoes other phosphorylation events that drive its ubiquitylation and ultimately lead to its degradation via the proteasome. Dashed line indicates that Dma1 and Dma2 ubiquitin ligases are involved, directly or indirectly, in Swe1 ubiquitylation.

So, Swe1 regulation is governed by complex pathways that are still partially unidentified. In particular, the involvement of the ubiquitylation pathway and the identity of the related ubiquitin ligase(s) involved in Swe1 degradation are unknown. A possible role in this control can be hypothesized for the functionally redundant budding yeast proteins Dma1 and Dma2, which belong to the same FHA-RING ubiquitin ligase family as *S. pombe* Dma1 and human Chfr and Rnf8. FHA domains are phosphothreonine-binding modules [24] frequently found in DNA repair and checkpoint proteins [25,26] and RING domains are typical of E3 ubiquitin ligases [26]. The presence of an FHA domain implies that one or more protein kinases function upstream of these proteins. An intact FHA domain is required for checkpoint function of all characterized FHA-RING ubiquitin ligases. All these proteins appear to control different aspects of the mitotic cell cycle, but several molecular details of their functions are still obscure [for review, see 27]. In particular, the *S. cerevisiae* Dma proteins are involved in mitotic checkpoints and contribute to control septin ring dynamics and cytokinesis by unknown mechanisms [28,29]. Moreover, *in vitro* ubiquitin ligase activity of Dma1 and Dma2 has been described [30], although their *in vivo* targets are still unknown.

380 Protein Phosphorylation in Human Health

Swe1 is subjected to multiple regulations that change its phosphorylation state (35-40 phosphorylated sites have been identified *in vivo*), subcellular localization and protein levels (Figure 1). During S phase, Swe1 accumulates in the nucleus where it is phosphorylated by Clb-Cdc28 before being exported to the cytoplams and then to the daughter side of the bud neck. Swe1 nuclear export is essential for its degradation in G2/M in fact a Swe1 variant that cannot be exported from the nucleus is largely stabilized [10]. Clb-Cdc28 phosphorylates Swe1 at multiple sites [11] and these events can occur in the nucleus, in the cytoplasm or at the bud neck. This multiphosphorylation by Cdc28-Clb has different roles: on one hand it promotes Swe1 activity generating a feedback loop, while on the other hand it promotes Swe1 degradation [12]. At the mother-bud neck, filaments of conserved proteins called septins (Cdc3, Cdc10, Cdc11, Cdc12 and Shs1 in *S. cerevisiae*) form a dynamic ring structure [13] that is essential for the recruitment of a number of proteins involved in the control of cell cycle progression [14,15]. Among these, the septin ring acts as a platform to recruit several Swe1 regulators, such as the Hsl1 protein kinase and its adaptor Hsl7, both essential for Swe1 localization to the bud neck and for its phosphorylation [7,9]. Hsl1, whose kinase activity requires assembled septins, undergoes autophosphorylation [8,16] and phosphorylates Hsl7 [9] but does not seem to phosphorylates Swe1 [17] although its kinase activity is required for Swe1 recruitment at the bud neck [18]. The fact that Hsl1 activation requires assembled septins ensures that Swe1 degradation does not begin until a bud has formed, thus providing a link between bud formation and entry into mitosis [18]. Also the PAK (p21-activated kinase) kinase Cla4 associates with the septin ring and is involved in Swe1 phosphorylation, probably during S phase [19]. Moreover Swe1 phosphorylation by Clb-Cdc28 promotes subsequent phosphorylation by the Polo-like kinase Cdc5; in fact the synergistic phosphorylation that can be observed *in vitro* on Swe1 by Clb2-Cdc28 and Cdc5 is the result of priming Swe1 by Cdc28, in which the resulting phosphorylated Swe1 becomes a better substrate for Cdc5 [20]. An additional level of Swe1 regulation involves the Cdc55 regulatory subunit of protein phosphatase PP2A, that has been implicated in its degradation since loss of Cdc55 function causes Swe1 stabilization [21]. However, how this control happens at the molecular level is not clear. In addition, a mathematical model for the morphogenesis checkpoint activation suggested that a subset of Swe1 phosphorylations could inhibit its activity whereas other phosphorylations could target Swe1 for degradation [22]. In any case, hyperphosphorylated Swe1 species are recognized by a still unidentified ubiquitin ligase and ubiquitylated [23]. Subsequently, Swe1 is degraded via the proteasome and this event allows mitotic entry [23]. However, how bud neck-localized Swe1 is targeted to degradation after phosphorylation is still obscure. There are Swe1 variants that do not undergo degradation although they show proper bud neck localization, phosphorylation and interaction with

known Swe1 regulators [23], indicating that still unknown Swe1 regulators exist.

So, Swe1 regulation is governed by complex pathways that are still partially unidentified. In particular, the involvement of the ubiquitylation pathway and the identity of the related ubiquitin ligase(s) involved in Swe1 degradation are unknown. A possible role in this control can be hypothesized for the functionally redundant budding yeast proteins Dma1 and Dma2, which belong to the same FHA-RING ubiquitin ligase family as *S. pombe* Dma1 and human Chfr and Rnf8. FHA domains are phosphothreonine-binding modules [24]

**Figure 1.** Model for Swe1 regulation. Swe1 shuttles from the nucleus to the cytoplasm and then it is translocated to the septin ring at the bud neck. These events are controlled through phosphorylation by Cdc28-Clb kinase. Once at the bud neck, Swe1 undergoes other phosphorylation events that drive its ubiquitylation and ultimately lead to its degradation via the proteasome. Dashed line indicates that Dma1 and Dma2 ubiquitin ligases are involved, directly or indirectly, in Swe1 ubiquitylation.

Recently, we provided genetic and biochemical evidence that Dma proteins are involved in promoting Swe1 ubiquitylation *in vivo* and contribute to the regulation of Swe1 stability by acting in a step following the recruitment of Swe1 to the bud neck and its phosphorylation [31]. Indeed, the lack of Dma proteins leads to accumulation of fully phosphorylated and bud neck localized Swe1. However, as *dma1 dma2* cells are viable while Swe1 degradation is essential for cell viability, other yet unidentified ubiquitin ligases likely ubiquitylate Swe1 during an unperturbed cell cycle. The Dma-dependent Swe1 down-regulation, whose lack does not significantly affect unperturbed cell cycle progression when the other Swe1 regulatory pathways are proficient, appears to be crucial for proper response to DNA replication stress.

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of Key Players in the Control of Cell Cycle Progression in *Saccharomyces cerevisiae* 383

mitotic exit. Kin4 is a non essential serine/threonine protein kinase that plays only a minor role in mitotic progression in normal growth conditions. But, importantly, Kin4 kinase participates in the SPOC and indeed it is essential to delay cell cycle progression of cells with a misaligned spindle. In case of spindle mispositioning, Kin4 maintains Bub2-Bfa1 GAP complex active through phosphorylation of Bfa1 on residues Ser150 and Ser180 [42], these

As already said, the subcellular localization of MEN and SPOC components is critical for their function, indeed the asymmetric distribution of MEN activators and inhibitors is one element that couples mitotic exit with correct nuclear migration. During an unperturbed cell cycle the Bub2-Bfa1 complex and Tem1 preferentially associates with the SPB that enters the daughter cell (dSPB) [35,37]. Instead the Lte1 protein, which positively regulates Tem1, is confined in the bud from the G1/S transition to telophase, when it diffuses throughout the cytoplasm of both mother cell and bud [37]. During anaphase, the SPB-associated Bub2- Bfa1 GAP complex keeps Tem1 inactive until the SPB and spindle enter the bud, where Tem1 is activated by its encounter with Lte1, thus promoting mitotic exit. Similarly to MEN factors, Kin4 binds the SPB in a Nud1 dependent manner [34,43]. During normal cell cycle progression, Kin4 localizes to the cortex of the mother cell by interaction of its Cterminal regulatory domain and this binding is a prerequisit for its loading on motherbound SPB (mSPB) during midanaphase [44]. When spindles is correctly oriented, Kin4 and Bub2-Bfa1 are asymmetrically localized to opposite SPBs. With anaphase onset, Bub2- Bfa1 then becomes inhibited by the Cdc5 kinase thus promoting mitotic exit. On the contrary, in case of spindle misalignment, Kin4 and Bub2-Bfa1 are brought together at both SPBs [45-48]; in these conditions Kin4 prevents Bfa1 phosphorylation by Cdc5, thereby inhibiting mitotic exit and this regulation is essential for survival of cells with a

So, Kin4 subcellular localization and activity are finely regulated during an unperturbed cell cycle and in response to spindle misalignment and these events involve its phosphorylation state (Figure 2). Kin4 phosphorylation state changes during the cell cycle: it is hyperphosphorylated during late stages of mitosis but is hypophosphorylated during Sphase and early mitosis [49]. At present, the relationship between Kin4 function, phosphorylation and localization is not fully understood, but at least three regulators of Kin4 have been identified: Lte1, the protein phosphatase PP2A-Rts1 and the bud neck kinase

Recent data indicate that Lte1 can regulate Kin4 by controlling its phosphorylation status [50]: Lte1 binds Kin4 and promotes its hyperphosphorylation and this event restricts Kin4 binding to the mSPB and prevents Kin4 that escapes the mother cell from associating with the dSPB. Importantly, this Lte1-mediated exclusion of Kin4 from the dSPB is essential for proper mitotic exit of cells with a correctly aligned spindle. Therefore, Lte1 promotes mitotic exit by inhibiting Kin4 activity at the dSPB. However, how this regulation happens remains to be determined. Indeed, Lte1 might regulate Kin4 activity itself, the Kin4 phosphatase PP2A-Rts1, the Kin4 kinase Elm1, other Kin4 kinases or the availability of Kin4 itself to be

events counteract Cdc5 action on Bfa1 and thereby inhibit mitotic exit.

misaligned spindle.

Elm1.

Collectively, the complex Swe1 regulation is an example of how phosphorylation events drive the fate of a protein. Indeed, Swe1 undergoes multiple phosphorylations that are crucial for its localization, its activity and its interaction with an E3 ubiquitin ligase that promote its degradation; moreover also some Swe1 regulators are regulated by phosphorylation, showing how this modification is largely involved in the control of cellular events.

## **3. The complex regulation of the protein kinase Kin4, a key player of the spindle position checkpoint**

The spindle position checkpoint (SPOC) is the important pathway that blocks mitotic exit and cytokinesis in case of mitotic spindle misalignment [32]. This pathway is crucial for budding yeast since the division site is determined early in the yeast cell cycle, in late G1 concomitantly with bud site selection, while the mitotic spindle is assembled after this event. So, in order to allow proper chromosome distribution between mother and daugher cell, the mitotic spindle must be correctly aligned with respect to the mother bud axis before mitotic exit. The SPOC monitors this event and arrests mitotic progression in case of spindle misalignment. The target of the SPOC is the GTPase Tem1, that acts at the top of the Mitotic Exit Network (MEN). The MEN is a signaling cascade of protein kinases that controls both exit from mitosis and cytokinesis. At the top of the pathway, the GTPase Tem1 activates Cdc15 kinase that leads to Dbf2/Dbf20 and Mob1 activation. The MEN ultimately activates the protein phosphatase Cdc14 that leads to Cdk1 inactivation, a key event for both mitotic exit and cytokinesis [33]. The localization of all these proteins is important for their activity. The spindle pole body (SPB) component Nud1 is the platform for most MEN components localization to the spindle poles during mitosis [34]. During every cell cycle, Tem1 is kept inactive until the mitotic spindle is properly aligned respect to the mother-bud axis, thus coupling mitotic exit with nuclear division [35-37]. Tem1 regulation is complex: it is positively regulated by Lte1 [35] and it is kept inactive by the dimeric GTPase-activating protein (GAP) Bub2-Bfa1, which is the target of two protein kinases, Cdc5 and Kin4 [38-41]. Cdc5 is the budding yeast Polo-like kinase, it plays multiple functions in mitosis and cytokinesis through phosphorylation of different substrates. Cdc5 inhibits Bfa1 by phosphorylation at the anaphase onset [40,41], thus promoting timely Tem1 activation and mitotic exit. Kin4 is a non essential serine/threonine protein kinase that plays only a minor role in mitotic progression in normal growth conditions. But, importantly, Kin4 kinase participates in the SPOC and indeed it is essential to delay cell cycle progression of cells with a misaligned spindle. In case of spindle mispositioning, Kin4 maintains Bub2-Bfa1 GAP complex active through phosphorylation of Bfa1 on residues Ser150 and Ser180 [42], these events counteract Cdc5 action on Bfa1 and thereby inhibit mitotic exit.

382 Protein Phosphorylation in Human Health

replication stress.

**spindle position checkpoint**

events.

Recently, we provided genetic and biochemical evidence that Dma proteins are involved in promoting Swe1 ubiquitylation *in vivo* and contribute to the regulation of Swe1 stability by acting in a step following the recruitment of Swe1 to the bud neck and its phosphorylation [31]. Indeed, the lack of Dma proteins leads to accumulation of fully phosphorylated and bud neck localized Swe1. However, as *dma1 dma2* cells are viable while Swe1 degradation is essential for cell viability, other yet unidentified ubiquitin ligases likely ubiquitylate Swe1 during an unperturbed cell cycle. The Dma-dependent Swe1 down-regulation, whose lack does not significantly affect unperturbed cell cycle progression when the other Swe1 regulatory pathways are proficient, appears to be crucial for proper response to DNA

Collectively, the complex Swe1 regulation is an example of how phosphorylation events drive the fate of a protein. Indeed, Swe1 undergoes multiple phosphorylations that are crucial for its localization, its activity and its interaction with an E3 ubiquitin ligase that promote its degradation; moreover also some Swe1 regulators are regulated by phosphorylation, showing how this modification is largely involved in the control of cellular

**3. The complex regulation of the protein kinase Kin4, a key player of the**

The spindle position checkpoint (SPOC) is the important pathway that blocks mitotic exit and cytokinesis in case of mitotic spindle misalignment [32]. This pathway is crucial for budding yeast since the division site is determined early in the yeast cell cycle, in late G1 concomitantly with bud site selection, while the mitotic spindle is assembled after this event. So, in order to allow proper chromosome distribution between mother and daugher cell, the mitotic spindle must be correctly aligned with respect to the mother bud axis before mitotic exit. The SPOC monitors this event and arrests mitotic progression in case of spindle misalignment. The target of the SPOC is the GTPase Tem1, that acts at the top of the Mitotic Exit Network (MEN). The MEN is a signaling cascade of protein kinases that controls both exit from mitosis and cytokinesis. At the top of the pathway, the GTPase Tem1 activates Cdc15 kinase that leads to Dbf2/Dbf20 and Mob1 activation. The MEN ultimately activates the protein phosphatase Cdc14 that leads to Cdk1 inactivation, a key event for both mitotic exit and cytokinesis [33]. The localization of all these proteins is important for their activity. The spindle pole body (SPB) component Nud1 is the platform for most MEN components localization to the spindle poles during mitosis [34]. During every cell cycle, Tem1 is kept inactive until the mitotic spindle is properly aligned respect to the mother-bud axis, thus coupling mitotic exit with nuclear division [35-37]. Tem1 regulation is complex: it is positively regulated by Lte1 [35] and it is kept inactive by the dimeric GTPase-activating protein (GAP) Bub2-Bfa1, which is the target of two protein kinases, Cdc5 and Kin4 [38-41]. Cdc5 is the budding yeast Polo-like kinase, it plays multiple functions in mitosis and cytokinesis through phosphorylation of different substrates. Cdc5 inhibits Bfa1 by phosphorylation at the anaphase onset [40,41], thus promoting timely Tem1 activation and As already said, the subcellular localization of MEN and SPOC components is critical for their function, indeed the asymmetric distribution of MEN activators and inhibitors is one element that couples mitotic exit with correct nuclear migration. During an unperturbed cell cycle the Bub2-Bfa1 complex and Tem1 preferentially associates with the SPB that enters the daughter cell (dSPB) [35,37]. Instead the Lte1 protein, which positively regulates Tem1, is confined in the bud from the G1/S transition to telophase, when it diffuses throughout the cytoplasm of both mother cell and bud [37]. During anaphase, the SPB-associated Bub2- Bfa1 GAP complex keeps Tem1 inactive until the SPB and spindle enter the bud, where Tem1 is activated by its encounter with Lte1, thus promoting mitotic exit. Similarly to MEN factors, Kin4 binds the SPB in a Nud1 dependent manner [34,43]. During normal cell cycle progression, Kin4 localizes to the cortex of the mother cell by interaction of its Cterminal regulatory domain and this binding is a prerequisit for its loading on motherbound SPB (mSPB) during midanaphase [44]. When spindles is correctly oriented, Kin4 and Bub2-Bfa1 are asymmetrically localized to opposite SPBs. With anaphase onset, Bub2- Bfa1 then becomes inhibited by the Cdc5 kinase thus promoting mitotic exit. On the contrary, in case of spindle misalignment, Kin4 and Bub2-Bfa1 are brought together at both SPBs [45-48]; in these conditions Kin4 prevents Bfa1 phosphorylation by Cdc5, thereby inhibiting mitotic exit and this regulation is essential for survival of cells with a misaligned spindle.

So, Kin4 subcellular localization and activity are finely regulated during an unperturbed cell cycle and in response to spindle misalignment and these events involve its phosphorylation state (Figure 2). Kin4 phosphorylation state changes during the cell cycle: it is hyperphosphorylated during late stages of mitosis but is hypophosphorylated during Sphase and early mitosis [49]. At present, the relationship between Kin4 function, phosphorylation and localization is not fully understood, but at least three regulators of Kin4 have been identified: Lte1, the protein phosphatase PP2A-Rts1 and the bud neck kinase Elm1.

Recent data indicate that Lte1 can regulate Kin4 by controlling its phosphorylation status [50]: Lte1 binds Kin4 and promotes its hyperphosphorylation and this event restricts Kin4 binding to the mSPB and prevents Kin4 that escapes the mother cell from associating with the dSPB. Importantly, this Lte1-mediated exclusion of Kin4 from the dSPB is essential for proper mitotic exit of cells with a correctly aligned spindle. Therefore, Lte1 promotes mitotic exit by inhibiting Kin4 activity at the dSPB. However, how this regulation happens remains to be determined. Indeed, Lte1 might regulate Kin4 activity itself, the Kin4 phosphatase PP2A-Rts1, the Kin4 kinase Elm1, other Kin4 kinases or the availability of Kin4 itself to be phosphorylated. So the SPOC function could be linked to the spatial restriction of the MEN regulators Kin4 and Lte1 and inhibition of Kin4 by Lte1 [50].

Protein Phosphorylation is an Important Tool to Change the Fate

of Key Players in the Control of Cell Cycle Progression in *Saccharomyces cerevisiae* 385

**Figure 2.** Model for Kin4 regulation. Kin4 is localized to the mother cell cortex and to the mother bound spindle pole body (SPB) when the mitotic spindle is correctly oriented. On the contrary, in case of spindle misalignment, Kin4 is present at both SPBs. The protein phosphatase PP2A-Rts1 is crucial for the proper localization of Kin4 to the mother cortex and SPBs both during the cell cycle and in response to spindle position defects. The protein kinase Elm1 activates Kin4 by direct phosphorylation and

**4. Budding yeast cytokinesis is controlled by several phosphoproteins**

Cytokinesis is the spatially and temporally regulated process by which, after chromosome segregation, eukaryotic cells divide their cytoplasm and membranes in order to produce two daughter cells. In budding yeast, the activity of the components of the citokinetic machinery is tightly controlled and their recruitment to the mother-bud neck follows a hieralchical order of assembly during the cell cycle. Most of these regulations are driven by

Cytokinesis completion is driven by complex and partially redundant pathways that regulate the assembly and contraction of an actomyosin ring (AMR) and the deposition of a trilaminary septum between mother and bud. In particular, the AMR is involved in constricting the plasma membrane at the division site to complete closure [57,58] and AMR

The first step towards cytokinesis is the assembly of a septin ring, which forms at the bud neck concomitantly with bud emergence as soon as cells enter S phase, and marks the

contraction is coupled to the centripetal growth of the primary septum (PS).

controls its localization.

phosphorylation and dephosphorylation events.

On the basis of the correlation between phosphorylation status and time of Kin4 activation during the cell cycle, it was hypothesized that dephosphorylated Kin4 might be its active form and that phosphatases that promote accumulation of this form would be required for Kin4 function. Recently, the protein phosphatase PP2A and its regulatory subunit Rts1 have been identified as Kin4 regulators. In particular, the phosphatase PP2A-Rts1 is required for Kin4 dephosphorylation during cell cycle entry and to maintain Kin4 in the dephosphorylated state during S phase and mitosis [49]. In addition, PP2A-Rts1 is crucial for proper localization of Kin4 to the mother cortex and SPBs both during the cell cycle and in response to spindle position defects [49]. The phosphatase does not appear to affect Kin4 kinase activity but instead it promotes its association with SPBs, which is essential for SPOC activity. So, PP2A-Rts1 functions upstream of Kin4, regulating its phosphorylation and localization during an unperturbed cell cycle and during SPOC activation, thus defining the phosphatase as a key regulator of SPOC function [49]. However, it is still unclear how Rts1 mobilizes Kin4 at the cortex and enables its association with SPBs, indeed it might dephosphorylate Kin4 or a Kin4 interactor at SPBs.

Another key player of Kin4 regulation is the bud neck-localized Elm1 kinase [51,52]. Elm1 has been previously implicated in septin organization, bud morphogenesis, bud neck integrity and cell cycle progression [53-55]. Recently it has been shown that Elm1 contributes to the SPOC by promoting Kin4 kinase activity. Indeed, Elm1 activates Kin4 by direct phosphorylation on Thr209, a residue in the activation loop, and on additional sites at the C terminus of Kin4 [51,52]. Kin4 phosphorylation by Elm1 is critical for its kinase activity and subsequent hyperphosphorylation [49,51,52]. At present, the molecular mechanism by which phosphorylation of Thr209 influences Kin4 catalytic function is unclear. Structural studies of kinases regulated by activation loop phosphorylation indicate that the loop might function as a "sensitive switch" that controls substrate binding triggered by phosphorylation-dependent conformational changes [56]. However, the lack of Kin4 kinase activity and hyperphosphorylation observed in *elm1* cells lead to reduced Kin4 binding to the mother cell cortex and SPOC deficiency. So, Elm1 function in the SPOC upstream of Kin4 by controlling its activity and localization.

Interestingly, we have recently described a new level of complexity in SPOC regulation. Indeed, we have implicated the E3 ubiquitin ligases Dma1 and Dma2 in the control of Elm1 bud neck localization [29]. Elm1 mislocalization in *dma1 dma2* cells results in reduced levels of Kin4 kinase activity and asymmetry of Bub2-Bfa1 at the spindle poles of mispositioned spindles. So, Dma1, Dma2, Elm1 and Kin4 are part of the same SPOC regulatory module, with Dma proteins controlling Elm1 localization, that is in turn required for full Kin4 activation. Importantly, it is worth noting that Dma1, Dma2, Elm1 and Kin4 all are required to prevent mitotic exit in response to spindle mispositioning but not to spindle depolymerization, differently from Bub2 and Bfa1 [28,39,52].

phosphorylated. So the SPOC function could be linked to the spatial restriction of the MEN

On the basis of the correlation between phosphorylation status and time of Kin4 activation during the cell cycle, it was hypothesized that dephosphorylated Kin4 might be its active form and that phosphatases that promote accumulation of this form would be required for Kin4 function. Recently, the protein phosphatase PP2A and its regulatory subunit Rts1 have been identified as Kin4 regulators. In particular, the phosphatase PP2A-Rts1 is required for Kin4 dephosphorylation during cell cycle entry and to maintain Kin4 in the dephosphorylated state during S phase and mitosis [49]. In addition, PP2A-Rts1 is crucial for proper localization of Kin4 to the mother cortex and SPBs both during the cell cycle and in response to spindle position defects [49]. The phosphatase does not appear to affect Kin4 kinase activity but instead it promotes its association with SPBs, which is essential for SPOC activity. So, PP2A-Rts1 functions upstream of Kin4, regulating its phosphorylation and localization during an unperturbed cell cycle and during SPOC activation, thus defining the phosphatase as a key regulator of SPOC function [49]. However, it is still unclear how Rts1 mobilizes Kin4 at the cortex and enables its association with SPBs, indeed it might

Another key player of Kin4 regulation is the bud neck-localized Elm1 kinase [51,52]. Elm1 has been previously implicated in septin organization, bud morphogenesis, bud neck integrity and cell cycle progression [53-55]. Recently it has been shown that Elm1 contributes to the SPOC by promoting Kin4 kinase activity. Indeed, Elm1 activates Kin4 by direct phosphorylation on Thr209, a residue in the activation loop, and on additional sites at the C terminus of Kin4 [51,52]. Kin4 phosphorylation by Elm1 is critical for its kinase activity and subsequent hyperphosphorylation [49,51,52]. At present, the molecular mechanism by which phosphorylation of Thr209 influences Kin4 catalytic function is unclear. Structural studies of kinases regulated by activation loop phosphorylation indicate that the loop might function as a "sensitive switch" that controls substrate binding triggered by phosphorylation-dependent conformational changes [56]. However, the lack of Kin4 kinase activity and hyperphosphorylation observed in *elm1* cells lead to reduced Kin4 binding to the mother cell cortex and SPOC deficiency. So, Elm1 function in the SPOC upstream of

Interestingly, we have recently described a new level of complexity in SPOC regulation. Indeed, we have implicated the E3 ubiquitin ligases Dma1 and Dma2 in the control of Elm1 bud neck localization [29]. Elm1 mislocalization in *dma1 dma2* cells results in reduced levels of Kin4 kinase activity and asymmetry of Bub2-Bfa1 at the spindle poles of mispositioned spindles. So, Dma1, Dma2, Elm1 and Kin4 are part of the same SPOC regulatory module, with Dma proteins controlling Elm1 localization, that is in turn required for full Kin4 activation. Importantly, it is worth noting that Dma1, Dma2, Elm1 and Kin4 all are required to prevent mitotic exit in response to spindle mispositioning but not to spindle

regulators Kin4 and Lte1 and inhibition of Kin4 by Lte1 [50].

dephosphorylate Kin4 or a Kin4 interactor at SPBs.

Kin4 by controlling its activity and localization.

depolymerization, differently from Bub2 and Bfa1 [28,39,52].

**Figure 2.** Model for Kin4 regulation. Kin4 is localized to the mother cell cortex and to the mother bound spindle pole body (SPB) when the mitotic spindle is correctly oriented. On the contrary, in case of spindle misalignment, Kin4 is present at both SPBs. The protein phosphatase PP2A-Rts1 is crucial for the proper localization of Kin4 to the mother cortex and SPBs both during the cell cycle and in response to spindle position defects. The protein kinase Elm1 activates Kin4 by direct phosphorylation and controls its localization.

## **4. Budding yeast cytokinesis is controlled by several phosphoproteins**

Cytokinesis is the spatially and temporally regulated process by which, after chromosome segregation, eukaryotic cells divide their cytoplasm and membranes in order to produce two daughter cells. In budding yeast, the activity of the components of the citokinetic machinery is tightly controlled and their recruitment to the mother-bud neck follows a hieralchical order of assembly during the cell cycle. Most of these regulations are driven by phosphorylation and dephosphorylation events.

Cytokinesis completion is driven by complex and partially redundant pathways that regulate the assembly and contraction of an actomyosin ring (AMR) and the deposition of a trilaminary septum between mother and bud. In particular, the AMR is involved in constricting the plasma membrane at the division site to complete closure [57,58] and AMR contraction is coupled to the centripetal growth of the primary septum (PS).

The first step towards cytokinesis is the assembly of a septin ring, which forms at the bud neck concomitantly with bud emergence as soon as cells enter S phase, and marks the

position where constriction between mother and daughter cell will take place at the end of mitosis. During S phase, the septin ring becomes an hourglass shaped structure that serves as a scaffold for recruiting other proteins to the bud neck, among which the type II myosin heavy chain Myo1 that forms a ring at the presumptive bud site during early S phase [57]. This Myo1 ring persists at the mother-bud neck until the end of anaphase, when a coincident ring of F-actin assembles and the resulting AMR eventually contracts, accomplishing septum formation. In late anaphase Hof1, Cyk3 and Inn1 are recruited sequentially to the bud neck. All these proteins are required to activate the chitin synthase Chs2 [59-61], which is in turn recruited to the division site after mitotic exit and is required to build the primary septum (PS) which is mostly made of chitin. Once the cytokinetic apparatus is fully assembled, AMR contraction, membrane invagination and PS synthesis all begin almost immediately. After completion of the PS, at either side of this structure, secondary septa (SS), which are made of the same components as the cell wall, i. e. glucans and mannan, are synthetized [62]. At this point, mother and daughter cells are connected by a trilaminar septum. Afterwards, cell separation is driven by the action of endochitinase, Cts1, and glucanases, Dse2 Dse4 Egt2, that degrade the PS from the daughter side [63,64]. Then mother and daughter cells separate permanently from each other leaving a disk of chitin, called "bud scar", on the mother cell surface.

Protein Phosphorylation is an Important Tool to Change the Fate

of Key Players in the Control of Cell Cycle Progression in *Saccharomyces cerevisiae* 387

It has instead been demonstrated that the Dbf2-Mob1 kinase, that appears at the division site just before AMR contraction [76,77], directly phosphorylates Hof1, a member of the F-BAR (Fes/CIP4 homology Bin/Amphiphysin/Rvsp) protein family conserved from yeast to mammals [78]. Hof1 protein levels, activity and localization are regulated by many phosphorylation events. Hof1 protein levels are regulated during the cell cycle, in particular Hof1 accumulates from G1/S phase, it disappears after mitotic exit, concomitantly with AMR contraction, and it remains unstable during the G1 phase of the following cell cycle. Hof1 colocalizes with the septin ring from S phase to late anaphase, then it interacts with the AMR before its contraction [79]. Efficient AMR contraction and cell separation are allowed by subsequent degradation of Hof1 by the action of the E3 ubiquitin ligase complex SCF (Skp, Cullin, F-box containing complex)/Grr1 [80] that can recognize its PEST (Proline, glutamin acid (E), Serine and Threonine) domain. Hof1 is directly phosphorylated by three kinases that act in concert to regulate its activity: Clb2-Cdc28, Cdc5 and Dbf2-Mob1 [81]. During mitosis, Hof1 undergoes Clb2-Cdc28 phosphorylation that does not seem to control its localization, but it primes Hof1 for subsequent phosphorylation by Cdc5 at Ser517. This event facilitates Hof1 binding to Mob1. The Dbf2-Mob1 kinase then phosphorylates several residues at both N- and C- terminal of Hof1 [81]. These modifications do not influence the physical interaction between Hof1 and Cyk3 or Inn1, instead they promote Hof1 release from the septin ring and localization to the AMR (Figure 3). There, phosphorylated Hof1

The third example is the regulation of the chitin synthase that builds the PS. Chs2 is an integral membrane protein that polymerizes chitin from its precursor UDP-N-acetylglucosamine. The chitin synthase 2 is synthetized in G2/M and accumulates in the endoplasmic reticulum (ER). The N-terminal and the central catalytic domain are located in the cytoplasm while the hydrophobic C-terminal domain is integrated into the ER membrane. During the metaphase to anaphase transition, Chs2 is phosphorylated in several Ser-Pro sites at its N-terminus exposed into the cytoplasm by Cdc28-Clb2 [82,83]. These phosphorylations events are important for Chs2 retention into the ER and therefore for its inibition. At the end of mitosis, MEN activation leads to the decrease of Cdc28-Clb2 activity and Chs2 can be exported from the ER and targeted to the division site [75,84-86]. The molecular mechanism of this release was unclear, but recent data indicate that Chs2 Nterminus is directly dephosphorylated by the protein phosphatase Cdc14 [86]. Then dephosphorylated Chs2 is traslocated to Golgi and, through secretory vescicoles, is delivered to the plasma membrane at the bud neck (Figure 3) [85,87]. There, Chs2 is active and promotes centripetally deposition of PS that occurs concomitantly with AMR contraction and membrane ingression. After PS completion Chs2 is removed from the bud neck by endocytosis and transferred to the vacuole where it is degraded by the action of the

protease Pep4 [87]. The signal that leads to Chs2 endocytosis is currently unknown.

modification in the control of a very important cell cycle process, the cytokinesis.

In summary, Cyk3, Hof1 and Chs2 regulations are good examples of how phosphorylation events can change the fate of a protein and underline the relevance of this posttranslational

promotes AMR contraction by an unknown mechanism.

The events leading to cytokinesis must be tightly controlled and coordinated with chromosome segregation and mitotic exit in order to ensure the genetic stability during cell growth and thereby the fate of daughter cells. Several pathways are able to regulate the last event of the cell cycle including the Mitotic Exit Network (MEN). The MEN seems to control not only the exit from mitosis but also the timing of cytokinesis. In fact, several MEN components localize to the division site after mitotic exit and they likely play a direct role in the regulation of cytokinesis. Indeed, as mitotic Cdk1 activity decrease, MEN kinases Cdc15, Dbf2/Dbf20, Mob1, Cdc5 and the protein phosphatase Cdc14 associate with the bud neck [65-69], in addition some MEN mutants fail to undergo cytokinesis [70,71]. Here we describe examples of regulation by phosphorylation and/or dephosphorylation during cytokinesis catalyzed by Cdc28-Clb2 and/or Dbf2-Mob1 kinases and also by Cdc14 phosphatase, and how these post-translational modifications can regulate the function of three important cytokinetic proteins, Cyk3, Hof1 and Chs2, in space and time.

Cyk3 is an SH3-domain protein and was isolated as high-copy suppressor of lethality in an *iqg1* strain [72] and in a *myo1* strain [73]. Cyk3 interacts with Hof1 and both cooperate to recruit Inn1 [61,74], that is essential for activation of chitin synthase and therefore for primary septum formation [60,61]. Overexpression of *CYK3* leads to an actomyosin independent recruitment of Inn1 to the bud neck [74], indicating that Cyk3 plays a central role in a rescue mechanism for cytokinesis in the absence of a functional AMR. Cyk3 activity and localization are positively regulated by phosphorylation events (Figure 3). Cyk3 total levels are constant throughout the cell cycle, but interestingly Cyk3 phosphorylated species begin to accumulate after mitotic exit and this leads to its recruitment to the bud neck. This phosphorylation event requires the MEN activity [75]. In particular, it has been proposed that Cyk3 is phosphorylated by Dbf2-Mob1 kinase but if it is its direct substrate has not been determined.

It has instead been demonstrated that the Dbf2-Mob1 kinase, that appears at the division site just before AMR contraction [76,77], directly phosphorylates Hof1, a member of the F-BAR (Fes/CIP4 homology Bin/Amphiphysin/Rvsp) protein family conserved from yeast to mammals [78]. Hof1 protein levels, activity and localization are regulated by many phosphorylation events. Hof1 protein levels are regulated during the cell cycle, in particular Hof1 accumulates from G1/S phase, it disappears after mitotic exit, concomitantly with AMR contraction, and it remains unstable during the G1 phase of the following cell cycle. Hof1 colocalizes with the septin ring from S phase to late anaphase, then it interacts with the AMR before its contraction [79]. Efficient AMR contraction and cell separation are allowed by subsequent degradation of Hof1 by the action of the E3 ubiquitin ligase complex SCF (Skp, Cullin, F-box containing complex)/Grr1 [80] that can recognize its PEST (Proline, glutamin acid (E), Serine and Threonine) domain. Hof1 is directly phosphorylated by three kinases that act in concert to regulate its activity: Clb2-Cdc28, Cdc5 and Dbf2-Mob1 [81]. During mitosis, Hof1 undergoes Clb2-Cdc28 phosphorylation that does not seem to control its localization, but it primes Hof1 for subsequent phosphorylation by Cdc5 at Ser517. This event facilitates Hof1 binding to Mob1. The Dbf2-Mob1 kinase then phosphorylates several residues at both N- and C- terminal of Hof1 [81]. These modifications do not influence the physical interaction between Hof1 and Cyk3 or Inn1, instead they promote Hof1 release from the septin ring and localization to the AMR (Figure 3). There, phosphorylated Hof1 promotes AMR contraction by an unknown mechanism.

386 Protein Phosphorylation in Human Health

chitin, called "bud scar", on the mother cell surface.

cytokinetic proteins, Cyk3, Hof1 and Chs2, in space and time.

strain [72] and in a *myo1*

*iqg1*

position where constriction between mother and daughter cell will take place at the end of mitosis. During S phase, the septin ring becomes an hourglass shaped structure that serves as a scaffold for recruiting other proteins to the bud neck, among which the type II myosin heavy chain Myo1 that forms a ring at the presumptive bud site during early S phase [57]. This Myo1 ring persists at the mother-bud neck until the end of anaphase, when a coincident ring of F-actin assembles and the resulting AMR eventually contracts, accomplishing septum formation. In late anaphase Hof1, Cyk3 and Inn1 are recruited sequentially to the bud neck. All these proteins are required to activate the chitin synthase Chs2 [59-61], which is in turn recruited to the division site after mitotic exit and is required to build the primary septum (PS) which is mostly made of chitin. Once the cytokinetic apparatus is fully assembled, AMR contraction, membrane invagination and PS synthesis all begin almost immediately. After completion of the PS, at either side of this structure, secondary septa (SS), which are made of the same components as the cell wall, i. e. glucans and mannan, are synthetized [62]. At this point, mother and daughter cells are connected by a trilaminar septum. Afterwards, cell separation is driven by the action of endochitinase, Cts1, and glucanases, Dse2 Dse4 Egt2, that degrade the PS from the daughter side [63,64]. Then mother and daughter cells separate permanently from each other leaving a disk of

The events leading to cytokinesis must be tightly controlled and coordinated with chromosome segregation and mitotic exit in order to ensure the genetic stability during cell growth and thereby the fate of daughter cells. Several pathways are able to regulate the last event of the cell cycle including the Mitotic Exit Network (MEN). The MEN seems to control not only the exit from mitosis but also the timing of cytokinesis. In fact, several MEN components localize to the division site after mitotic exit and they likely play a direct role in the regulation of cytokinesis. Indeed, as mitotic Cdk1 activity decrease, MEN kinases Cdc15, Dbf2/Dbf20, Mob1, Cdc5 and the protein phosphatase Cdc14 associate with the bud neck [65-69], in addition some MEN mutants fail to undergo cytokinesis [70,71]. Here we describe examples of regulation by phosphorylation and/or dephosphorylation during cytokinesis catalyzed by Cdc28-Clb2 and/or Dbf2-Mob1 kinases and also by Cdc14 phosphatase, and how these post-translational modifications can regulate the function of three important

Cyk3 is an SH3-domain protein and was isolated as high-copy suppressor of lethality in an

recruit Inn1 [61,74], that is essential for activation of chitin synthase and therefore for primary septum formation [60,61]. Overexpression of *CYK3* leads to an actomyosin independent recruitment of Inn1 to the bud neck [74], indicating that Cyk3 plays a central role in a rescue mechanism for cytokinesis in the absence of a functional AMR. Cyk3 activity and localization are positively regulated by phosphorylation events (Figure 3). Cyk3 total levels are constant throughout the cell cycle, but interestingly Cyk3 phosphorylated species begin to accumulate after mitotic exit and this leads to its recruitment to the bud neck. This phosphorylation event requires the MEN activity [75]. In particular, it has been proposed that Cyk3 is phosphorylated by Dbf2-Mob1 kinase but if it is its direct substrate has not been determined.

strain [73]. Cyk3 interacts with Hof1 and both cooperate to

The third example is the regulation of the chitin synthase that builds the PS. Chs2 is an integral membrane protein that polymerizes chitin from its precursor UDP-N-acetylglucosamine. The chitin synthase 2 is synthetized in G2/M and accumulates in the endoplasmic reticulum (ER). The N-terminal and the central catalytic domain are located in the cytoplasm while the hydrophobic C-terminal domain is integrated into the ER membrane. During the metaphase to anaphase transition, Chs2 is phosphorylated in several Ser-Pro sites at its N-terminus exposed into the cytoplasm by Cdc28-Clb2 [82,83]. These phosphorylations events are important for Chs2 retention into the ER and therefore for its inibition. At the end of mitosis, MEN activation leads to the decrease of Cdc28-Clb2 activity and Chs2 can be exported from the ER and targeted to the division site [75,84-86]. The molecular mechanism of this release was unclear, but recent data indicate that Chs2 Nterminus is directly dephosphorylated by the protein phosphatase Cdc14 [86]. Then dephosphorylated Chs2 is traslocated to Golgi and, through secretory vescicoles, is delivered to the plasma membrane at the bud neck (Figure 3) [85,87]. There, Chs2 is active and promotes centripetally deposition of PS that occurs concomitantly with AMR contraction and membrane ingression. After PS completion Chs2 is removed from the bud neck by endocytosis and transferred to the vacuole where it is degraded by the action of the protease Pep4 [87]. The signal that leads to Chs2 endocytosis is currently unknown.

In summary, Cyk3, Hof1 and Chs2 regulations are good examples of how phosphorylation events can change the fate of a protein and underline the relevance of this posttranslational modification in the control of a very important cell cycle process, the cytokinesis.

Protein Phosphorylation is an Important Tool to Change the Fate

of Key Players in the Control of Cell Cycle Progression in *Saccharomyces cerevisiae* 389

In addition, a protein can be modified by the addition of a single phosphate group or by multiple phosphates, by a single protein kinase or by multiple kinases. Multisite phosphorylation is a strategy that enables two or more effects to operate in the same protein. Indeed, some phosphorylation events "prime" the protein in order to be phosphorylated by another kinase that acts subsequently in the same cellular compartment or in another location. The protein kinase Swe1 is a very good example of this kind of regulation, in fact several kinases act sequentially leading to the accumulation of hyperphosphorylated bud neck localized Swe1. Also the cytokinetic proteins Hof1 and Chs2 are the target of at least two different protein kinases that change Hof1 and Chs2 activity and localization. Alternatively, a phosphorylation event can inhibit the phosphorylation of other residues on the same protein. For example, Bfa1 phosphorylation by Kin4 inhibits phosphate group

Another important issue is the crosstalk between phosphorylation and other posttranslational modifications. Crosstalks can be positive or negative, thus promoting or inhibiting the subsequent modification. About phosphorylation and ubiquitylation, an increasing number of phosphoproteins are then ubiquitylated. Both Hof1 and Swe1 are hyperphosphorylated and afterwards ubiquitylated, subsequently they are targeted to degradation via the proteasome. A very interesting issue is how the specificity is determined. Indeed, phosphorylated residues of serine, threonine and tyrosine recognition by phosphobinding domains depends on the sequence of amino acids immediately around the phosphorylated residue, whereas recognition of monoUb by ubiquitin binding domains does not seem to be influenced by the primary sequence bearing the ubiquitinated lysine.

Even if there are several open questions regarding the molecular mechanisms that control the phosphoproteins that we have described, they are good examples of how the

*Università degli Studi di Milano-Bicocca, Dipartimento di Biotecnologie e Bioscienze, Milano, Italy*

Roberta Fraschini's research is supported by grants from PRIN (Progetti di Ricerca di Interesse Nazionale) 2008. C. C. is supported by a fellowship from Fondazione Confalonieri.

[1] Morgan DO (2007) The Cell Cycle: Principles of Control. Oxford University Press.

addition by the polo-like kinase Cdc5 and so keeps Bfa1 active.

phosphorylation event can change the fate of a protein.

, Erica Raspelli† and Corinne Cassani

**Author details**

Roberta Fraschini\*

**6. References**

 Corresponding Author † Equally Contributing Authors

 \*

**Acknowledgement**

**Figure 3.** Model for Cyk3, Hof1 and Chs2 regulation. Cyk3 is likely phosphorylated by Dbf2-Mob1 kinase (dashed line) and recruited to the bud neck. Septin bound Hof1 is "primed" by Cdc28-Clb2 phosphorylation and then it is phosphorylated by polo-like kinase Cdc5 and by Dbf2-Mob1 kinase. These modifications promote Hof1 release from the septin ring and localization to the actomyosin ring. Chs2 accumulates in the endoplasmic reticulum (ER) with its N-terminus exposed into the cytoplasm, this tail is phosphorylated by Cdc28-Clb2. These phosphorylations events are important for Chs2 retention into the ER. At the end of mitosis, Chs2 N-terminus is directly dephosphorylated by the protein phosphatase Cdc14, then dephosphorylated Chs2 is traslocated to Golgi and, through secretory vescicoles, is delivered to the plasma membrane at the bud neck.

## **5. Conclusion**

The phosphorylation of a protein is a simple mechanism that alters its conformation, and so its ability to function, in a reversibile way. As we can learn from the examples of protein regulation that we have focused on, phosphorylation is a flexible mechanism that regulates the target protein in several ways. Indeed, phosphorylation is not simply used to switch the activity of a protein on or off, but can have many additional roles. It can influence its ability to form complexes with other proteins, it can affect the rate at which a protein is degraded or its ability to localize to a particular subcellular location. For example, phosphorylation events on the protein kinases Swe1 and Kin4, and on the cytokinetic proteins Cyk3, Hof1 and Chs2 lead to change in their localization.

The action of kinases is counteracted by phosphatases and both controls are essential to determine the phosphorylation state of the target proteins. The balance of phosphorylation and dephosphorylation can also be critical in determining the strength and duration of the response. Therefore, kinases and phosphatases must be regulated spatiotemporally in order to obtain the proper cellular response.

In addition, a protein can be modified by the addition of a single phosphate group or by multiple phosphates, by a single protein kinase or by multiple kinases. Multisite phosphorylation is a strategy that enables two or more effects to operate in the same protein. Indeed, some phosphorylation events "prime" the protein in order to be phosphorylated by another kinase that acts subsequently in the same cellular compartment or in another location. The protein kinase Swe1 is a very good example of this kind of regulation, in fact several kinases act sequentially leading to the accumulation of hyperphosphorylated bud neck localized Swe1. Also the cytokinetic proteins Hof1 and Chs2 are the target of at least two different protein kinases that change Hof1 and Chs2 activity and localization. Alternatively, a phosphorylation event can inhibit the phosphorylation of other residues on the same protein. For example, Bfa1 phosphorylation by Kin4 inhibits phosphate group addition by the polo-like kinase Cdc5 and so keeps Bfa1 active.

Another important issue is the crosstalk between phosphorylation and other posttranslational modifications. Crosstalks can be positive or negative, thus promoting or inhibiting the subsequent modification. About phosphorylation and ubiquitylation, an increasing number of phosphoproteins are then ubiquitylated. Both Hof1 and Swe1 are hyperphosphorylated and afterwards ubiquitylated, subsequently they are targeted to degradation via the proteasome. A very interesting issue is how the specificity is determined. Indeed, phosphorylated residues of serine, threonine and tyrosine recognition by phosphobinding domains depends on the sequence of amino acids immediately around the phosphorylated residue, whereas recognition of monoUb by ubiquitin binding domains does not seem to be influenced by the primary sequence bearing the ubiquitinated lysine.

Even if there are several open questions regarding the molecular mechanisms that control the phosphoproteins that we have described, they are good examples of how the phosphorylation event can change the fate of a protein.

## **Author details**

388 Protein Phosphorylation in Human Health

**Figure 3.** Model for Cyk3, Hof1 and Chs2 regulation. Cyk3 is likely phosphorylated by Dbf2-Mob1 kinase (dashed line) and recruited to the bud neck. Septin bound Hof1 is "primed" by Cdc28-Clb2 phosphorylation and then it is phosphorylated by polo-like kinase Cdc5 and by Dbf2-Mob1 kinase. These modifications promote Hof1 release from the septin ring and localization to the actomyosin ring. Chs2 accumulates in the endoplasmic reticulum (ER) with its N-terminus exposed into the cytoplasm, this tail is phosphorylated by Cdc28-Clb2. These phosphorylations events are important for Chs2 retention into the ER. At the end of mitosis, Chs2 N-terminus is directly dephosphorylated by the protein phosphatase Cdc14, then dephosphorylated Chs2 is traslocated to Golgi and, through secretory

The phosphorylation of a protein is a simple mechanism that alters its conformation, and so its ability to function, in a reversibile way. As we can learn from the examples of protein regulation that we have focused on, phosphorylation is a flexible mechanism that regulates the target protein in several ways. Indeed, phosphorylation is not simply used to switch the activity of a protein on or off, but can have many additional roles. It can influence its ability to form complexes with other proteins, it can affect the rate at which a protein is degraded or its ability to localize to a particular subcellular location. For example, phosphorylation events on the protein kinases Swe1 and Kin4, and on the cytokinetic proteins Cyk3, Hof1

The action of kinases is counteracted by phosphatases and both controls are essential to determine the phosphorylation state of the target proteins. The balance of phosphorylation and dephosphorylation can also be critical in determining the strength and duration of the response. Therefore, kinases and phosphatases must be regulated spatiotemporally in order