Polyoma and SV40 proteins differentially regulate PP2A to activate ...

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Dec 19, 2006 - SV40 small T-antigens influence PP2A to activate discrete cellular signaling pathways involved in growth control. AKT MAP kinase cascade ...
Polyoma and SV40 proteins differentially regulate PP2A to activate distinct cellular signaling pathways involved in growth control Pablo Rodriguez-Viciana, Crista Collins, and Mike Fried* Cancer Research Institute, University of California, San Francisco, CA 94143 Communicated by James E. Cleaver, University of California, San Francisco, CA, October 24, 2006 (received for review September 5, 2006)

Binding of Src family kinases to membrane-associated polyoma virus middle T-antigen (PyMT) can result in the phosphorylation of PyMT tyrosine 250, which serves as a docking site for the binding of Shc and subsequent activation of the Raf-MEK-ERK (MAP) kinase cascade. In a screen for PyMT variants that could not activate the ARF tumor suppressor, we isolated a cytoplasmic nontransforming mutant (MTA) that encoded a C-terminal truncated form of the PyMT protein. Surprisingly, MTA was able to strongly activate the MAP kinase pathway in the absence of Src family kinase and Shc binding. Interestingly, the polyoma small T-antigen (PyST), which shares with MTA both partial amino acid sequence homology and cellular location, also activates the MAP kinase cascade. Activation of the MAP kinase cascade by both MTA and PyST has been demonstrated to be PP2A-dependent. Neither MTA nor PyST activate the phosphorylation of AKT. The SV40 small T-antigen, which is similar to PyST in containing a J domain and in binding to the PP2A AC dimer, does not activate the MAP kinase cascade, but does stimulate phosphorylation of AKT in a PP2A-dependent manner. These findings highlight a novel role of PP2A in stimulating the MAP kinase cascade and indicate that the similar polyoma and SV40 small T-antigens influence PP2A to activate discrete cellular signaling pathways involved in growth control. AKT 兩 MAP kinase cascade 兩 polyoma virus T-antigens 兩 SV40 small T-antigen

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ctivating oncogenes overcome normal cell regulatory control systems and stimulate growth signaling pathways leading to unregulated cell division. The polyoma virus (Py) early region specifies three proteins, large T-antigen (PyLT), middle T-antigen (PyMT), and small T-antigen (PyST), by differential splicing of mRNA derived from the same genomic DNA sequence (1). The membrane-bound PyMT is a potent activating oncogene, which has no intrinsic enzyme activity but acts as a scaffolding protein and binds and modulates the activities of a number of important cellular signaling proteins (1, 2). Studies with PyMT have provided great insight into a number of critical cellular signaling pathways involved in growth control (3). Membrane-bound PyMT binds and activates the cellular Src tyrosine kinase family resulting in phosphorylation of a number of important PyMT tyrosine residues (1, 2) (Figs. 1 and 2). Phosphorylation of PyMT tyrosine 315 (Y315) results in the binding of the PI3-kinase regulatory p85 subunit and subsequent activation of PI3-kinase activity, resulting in the phosphorylation of a number of cellular targets, including AKT (Figs. 1 and 2). Phosphorylation of PyMT tyrosine 250 (Y250) results in the binding of the Shc family of cellular proteins. Bound Shc associates with the adapter molecule Grb2, which binds to the Sos guanine nucleotide exchange factor, resulting in the activation of Ras. Ras activation leads to the activation of the Raf-MAP kinase pathway resulting in the phosphorylation of MEK and ERK (Fig. 1). Phosphorylation of PyMT tyrosine 322 (Y322) results in the binding of PLC␥ and activation of its associated signal transduction pathways (Fig. 2). 19290 –19295 兩 PNAS 兩 December 19, 2006 兩 vol. 103 兩 no. 51

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Motility, Differentiation, Proliferation, Survival Proliferation Fig. 1. PyMT Shc and PI3-kinase signaling. A diagrammatic representation of Shc and PI3-kinase (PI3K) pathways stimulated by membrane-bound PyMT is shown. Association of Src family kinases with membrane-bound PyMT can result in the phosphorylation of PyMT tyrosines 250 (Y250) and 315 (Y315). Phosphorylation of PyMT Y250 can serve as a docking site for Shc, which recruits the Grb2 adaptor protein, which binds the Sos guanine nucleotide exchange factor resulting in the activation of Ras. Ras activation can lead to the activation of the Raf-Map kinase cascade, resulting in the phosphorylation of MEK and ERK. Phosphorylation of PyMT tyrosine 315 (Y315) results in the binding of the PI3-kinase regulatory p85 subunit and subsequent activation of PI3-kinase activity resulting in the phosphorylation of a number of cellular targets including AKT.

PyMT also induces ARF, and consequently, p53, as a result of the inappropriate induction of one or more of the growth stimulatory pathways it activates (4, 5). PyMT, as well as the PyST with which it shares extensive amino acid homology, contains a PP2A binding domain that allows it to mimic the cellular PP2A regulatory B subunit and bind to the core PP2A dimer composed of the A and C subunits (6, 7) (Fig. 2). The binding of PP2A subunits A and C to PyMT is required for the stable association of the Src family kinases, and in the absence of the binding of these PP2A subunits, none of the PyMT tyrosines are phosphorylated and the cellular growth signal transduction pathways are not activated (1). In Author contributions: P.R.-V. and M.F. designed research; P.R.-V., C.C., and M.F. performed research; P.R.-V. and M.F. contributed new reagents/analytic tools; P.R.-V. and M.F. analyzed data; and P.R.-V. and M.F. wrote the paper. The authors declare no conflict of interest. Abbreviations: Py, polyoma virus; PyLT, Py large T-antigen; PyMT, Py middle T-antigen; PyST, Py small T-antigen; SVST, SV40 small T-antigen. *To whom correspondence should be addressed at: Cancer Research Institute, University of California, 2340 Sutter Street, San Francisco, CA 94143-0128. E-mail: [email protected]. © 2006 by The National Academy of Sciences of the USA

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PyST in activating the MAP kinase pathway through regulation of PP2A. We also observe that MTA and PyST differ from SV40 small T-antigen (SVST), which activates the PI3-kinase pathway but not the MAP kinase pathway. Results

Fig. 2. PyMT binding sites. A diagrammatic representation of the membrane localized PyMT wild type (Top), the cytoplasmic MTA (Middle), and the nuclear MT11 (Bottom) proteins are shown. The location of the binding sites for Hsc70 (J domain), PP2A A and C subunits (PP2A domain), Src family kinases, 14-3-3, Shc, PI3-kinase, and PLC␥, as well as the position of the membrane localization signal on the 421-aa PyMT wild-type protein, are shown. PyMT shares its N-terminal 191 aa (shown in gray) with PyST, which contains the J and PP2A domains. The MTA frameshift protein contains the first 337 aa of PyMT attached to 6 aa from a small ORF of the 3⬘ untranslated region of the PyST (stippled) at its C terminus (8). The MT11 frameshift protein contains the N-terminal 338 aa of PyMT attached to 85 aa of PyLT (containing nuclear localization sequence) (diagonal crosshatch) and 4 aa of vector sequence (horizontal crosshatch) at its C terminus (8).

addition, PyMT can bind the Hsc70 and the 14-3-3 cellular proteins (1, 2) (Fig. 2). In a previous study, we used a screen to isolate untransformed REF52 cell clones containing integrated expressed PyMT mutant sequences, which could not activate the ARF-p53 pathway and block cell division (8). The integrated PyMT DNA sequences in two of these REF52 cell clone were found to be altered at a mutational DNA hotspot region containing 9 consecutive cytosines resulting in different frameshift mutations. As a consequence of either a loss (MTA) or gain (MT11) of one cytosine, each of the encoded PyMT mutant proteins frameshifted into one of the two alternative Py reading frames (8). This results in the loss of the PyMT C-terminal membrane binding region and the subsequent mislocalization of the PyMT mutant proteins to either the cytoplasm (MTA) or the nucleus (MT11) (8). The organization of the MTA and MT11 mutant PyMT proteins relative to the wild-type PyMT protein is shown in Fig. 2. In this work, we find that the MTA PyMT mutant resembles

PyMT proteins activate the ARF-p53 pathway or induce neoplastic transformation (8). To gain some insight into which PyMT cellular binding proteins might be involved in the induction of the ARF-p53 pathway and/or neoplastic transformation, we sought to determine which cellular pathways were or were not activated by the variant MTA and MT11 PyMT proteins. The wild-type PyMT protein binds to and activates Src family kinases in the membrane, resulting in the phosphorylation of a number of PyMT tyrosines. These phospho-tyrosines serve as docking sites for various proteins, which upon binding to PyMT lead to the activation of a number of important cellular signaling pathways (Figs. 1 and 2) (1, 2). The PyMT Src-family binding region is present in both the MTA- and MT11-truncated PyMT proteins (Fig. 2), but neither of these mutant proteins is located in the membrane (8) and would not be expected to be associated with members of the membrane-localized Src kinase family as has been observed for other PyMT mutants mutated in their membrane localization region (9, 10). To assess whether MTA and MT11 mutant PyMT proteins are affected in their ability to associate with Src-family kinases, we immunoprecipitated the various PyMT proteins and measured their associated kinase activity by performing in vitro kinase assays, using the PyMT proteins as the substrate (Fig. 3). Because wild-type PyMT alone will activate ARF and consequently p53 in normal REF52 cells resulting in a block to cell division (4), in this and in following experiments we used dividing PyMT transformed REF52 cells in which p53 is inactivated by the presence of a dominant negative p53 (PyMT DNp53REF52) as a positive control. The results (Fig. 3) show that a very small amount (2–5%) of kinase activity is associated with the MTA- and MT11-altered PyMT proteins compared with wild-type PyMT. Thus, the MTAand MT11-PyMT proteins are severely inhibited in their associated Src family kinase activity, as has been found for other PyMT mutants that are impaired in their membrane localization (9, 10). We cannot rule out that the small amount of kinase activity we detect is not the result of association of the mutant PyMT with a Src kinase family member in the cell extract after breaking open the cells.

Fig. 3. MTA and MT11 have reduced MT-associated in vitro kinase activity. (Left) Wild-type PyMT proteins from PyE REF52 cells and PyMT DNp53REF52 cells, and the mutant PyMT proteins from MT11 and MTA REF52 cells after immunoprecipitation with the 762 antibody and Western blotting with the PyC antibody. (Right) Autoradiogram of the same PyMT immunoprecipitated species shown in Left, after incubation in the presence of ␥-ATP to detect MT-associated kinase activity. The wild-type PyMT from PyE REF52 and PyMT DNp53REF52 cells contain an associated kinase activity, which is able to phosphorylate the PyMT protein, whereas little kinase activity (2–5%) is found associated with the mutant proteins from the MT11 and MTA cells.

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The Cytoplasmic MTA PyMT Protein and the Nuclear MT11 PyMT Retain Minimal in Vitro Kinase Activity. Neither the MTA nor the MT11

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PP2A 35kd C Subunit Fig. 4. Interaction of PyMT proteins with Shc and PP2A. Wild-type and mutant PyMT proteins were immunoprecipitated (IP) from PyMT DNp53REF52 (MT REF52) and MTA and MT11 REF52 cells with the 762 anti-Py antibody, gel fractionated, and probed with different antibodies. (Top) Various IPs probed with PyC anti-PY antibody and the locations of the different PyMT proteins. (Middle) Fractionated IP proteins probed with anti-Shc antibody. The location of the 66kd, 52kd, and 42kd Shc species are indicated. (Bottom) Fractionated IP proteins probed with a mixture of antibodies against the PP2A A and C subunits. The location of the PP2A 65kd A and 35kd C subunits are indicated. The location of the nonspecific cross-reactivity to the antibody heavy chain (Ab) used in the immunoprecipitation is indicated in Middle and Bottom.

Analysis of the Cellular Pathways Activated by the Altered PyMT Species in Untransformed MTA and MT11 Cells. Retained in the

PyMT amino acids sequences present in both the MTA and MT11 mutant PyMT proteins are the two important PyMT tyrosines, Y250 and Y315 (Fig. 2). Phosphorylated Y250 and Y315, serve as docking sites for Shc family proteins and the PI3-kinase p85 regulatory subunit respectively (Figs. 1 and 2). Shc proteins bind to the adapter molecule Grb2, which associates with the Sos guanine nucleotide exchange factor. Binding of Shc to PyMT results in the translocation of Sos to the membrane and activation of Ras. Ras then activates Raf kinase leading to the sequential phosphorylation and activation of MEK (P-MEK) and ERK (P-ERK) (Fig. 1). Binding of the p85 regulatory subunit to PyMT phosphorylated at Y315 can lead to activation of the PI3-kinase pathway, resulting in the phosphorylation of a number of cellular targets including AKT (P-AKT) (Fig. 1). The abilities of MTA and MT11 mutant proteins to associate with Shc proteins were assayed by immunoprecipitation followed by Western blot analysis (Fig. 4). Whereas Shc proteins could be detected bound to wild-type PyMT, no Shc was detected complexed with either the MTA- or MT11-mutant PyMT proteins. Thus, the lack of association of the MTA and MT11 mutant PyMT proteins with the Src kinases correlates with their inability to interact with Shc proteins. On the other hand, the MTA and MT11 mutant proteins are similar to wild-type PyMT in their ability to interact with the PP2A A and C subunits as both of these PP2A subunits are readily detected in immunoprecipitates of MTA, MT11, and wild-type PyMT proteins (Fig. 4). MTA Activates the MAP Kinase Pathway in a PP2A-Dependent Manner.

We assessed the activation state of the MAP kinase and PI3kinase pathways in cells expressing the various PyMT proteins by Western blot analysis on cell lysates using phospho-specific antibodies to ERK, MEK, and AKT (Fig. 5). As expected, increased levels of P-ERK, P-MEK, and P-AKT were detected in REF52 cells transformed by wild-type PyMT. Unlike wildtype PyMT-expressing cells, MTA- or MT11-expressing cells did 19292 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0609343103

not contain elevated levels of P-AKT. MT11 cells were also defective in activating the MAP kinase pathway. Surprisingly, however, MTA-expressing cells did show high levels of phosphorylated MEK and ERK (Fig. 5A). To rule out the possibility that these results were a peculiarity of the MTA and MT11 cell lines being studied, retroviruses containing the MTA and MT11 mutant PyMT sequences were generated and used to infect wild-type REF52 cells. Similar levels of P-AKT, P-ERK, and P-MEK, as expressed in the MTA and MT11 REF52 cell lines, were found in REF52 cells 6 days after retrovirus infection (data not shown). Interestingly, levels of P-ERK and P-MEK were consistently higher in cells containing the MTA protein than in cells containing wild-type PyMT, both in the cell lines and in retrovirus infected cells (Fig. 5). Thus, despite its failure to interact with Shc (Fig. 4), the MTA-mutant PyMT protein can strongly activate the MAP kinase pathway (Fig. 5A). To assess the possibility that residual Shc binding below the level of detection of our assay might be responsible for MAP kinase pathway activation, we mutated the PyMT tyrosine that serves as a Shc-binding site to phenylalanine (Y250F) in the MTA-mutant protein. When expressed in cells, the MTA Y250F PyMT protein is still able to activate ERK phosphorylation (Fig. 5B), indicating that MTA can activate the MAP kinase pathway by a mechanism independent of Shc binding. Strikingly, a MTA-mutant protein that is defective in binding to PP2A subunits (MTA PP2A-) as the result of an insertion of alanine and leucine residues at PyMT amino acid 107 (11) is unable to activate the MAP kinase pathway (Fig. 5B). As MEK and ERK were activated in MTA expressing cells (Figs. 5 A and B), we evaluated whether the Raf was also activated. We tested for Raf activation by assessing whether Raf was phosphorylated at activating serine 338 (12–14). As can be seen in Fig. 5C, phosphorylation of Raf serine 338 can be detected in REF52 cells transformed by wild-type PyMT and in MTA cells but not in untransformed REF52 cells or MT11 cells. Thus, three of the components of the MAP kinase pathway, Raf, MEK, and ERK, are activated in MTA cells but not MT11 cells. Rodriguez-Viciana et al.

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Total Raf1 Fig. 5. MTA PyMT activates the MAP kinase pathway in a PP2A-dependent manner. (A) ERK and MEK, but not AKT, phosphorylation is stimulated in MTA REF52 cells. Gel fractionated extracts derived from REF52, DNp53REF52, PyMT transformed DNp53REF52, MT11, and MTA REF52 cells were probed with phospho-specific antibodies to AKT, ERK, and MEK. The total amount of ERK and MEK in the different extracts is shown. (B) Effect of inactivation of the PyMT Shc or PP2A-binding sites on the stimulation of phospho-ERK in MTAexpressing cells. REF52 cells were infected with retroviruses containing either the wild-type MTA sequence (MTA WT) or MTA sequence mutated in the PP2A binding domain (MTA PP2A-) or mutated in the Shc binding tyrosine 250 (MTA Y250F). ERK phosphorylation was measured in extracts from each infected cell population using phospho-specific antibodies after gel fractionation (Upper). The filter was reprobed with a PyMT antibody (Lower) to show expression levels of the different MTA proteins. (C) Phosphorylation of the Raf1 serine 388 activating site is stimulated in MTA cells. Phosphorylation of Raf1 at the activating serine 338 site (P-S338) was measured by using a phospho-specific antibody after gel fractionation of Raf1 immunoprecipitates from extracts of MTA, MT11, DNp53REF52, DNp53REF52 cells treated with EGF and PyMT transformed DNp53REF52 cells. In Lower, total Raf1 in each sample is shown as a loading control.

The MTA Protein Mimics PyST. PyMT contains the same N-terminal 191 amino acids as PyST (see Fig. 2). Within this common region is the PP2A binding domain (Fig. 2). Nevertheless, the two proteins have different cellular localizations. Whereas PyMT is membrane associated, PyST is found in both the cytoplasm and the nucleus. Both the cytoplasmic MTA protein and the nuclear MT11 protein both contain 191 of the 195 of the PyST amino acids (Fig. 2) and bind PP2A subunits A and C (Fig. 4). Thus, we sought to determine whether the effect of PyST on the PI3kinase and MAP kinase pathways was similar to those we observed for the MTA- and/or MT11-mutant PyMT proteins. REF52 cells were infected with retroviruses containing wildtype PyST or PyST containing mutations in either the J domain or the PP2A domain. The activation of the MAP kinase pathway and the PI3K pathway in the infected cells were analyzed by Western blot analysis with phospho-specific antibodies (Fig. 6A). Wild-type PyST, like MTA, strongly stimulates MEK and ERK but not AKT, phosphorylation. In fact, expression of PP2Acompetent PyST consistently leads to a decrease in the levels of basal phosphorylated AKT (see Fig. 6A). Mutation in the J domain, which binds the Hsc70 heat shock protein, does not

BJ fibroblasts

Fig. 6. PyST activates the MAP kinase pathway whereas SVST activates the PI3K pathway. (A) PyST stimulates ERK phosphorylation in a PP2A-dependent manner. REF52 cells were infected with retroviruses containing the MTA sequence or PyST wild type, PyST mutated in the PP2A (PyST PP2A-) or J (PyST J Domain-) domain, or no insert (empty). Extracts from the infected cells were gel fractionated and assessed for phosphorylated AKT (P-AKT) or phosphorylated ERK (P-ERK) using phospho-specific antibodies. The amount of total ERK in each extract is shown. (B) PyST stimulates PP2A-dependent ERK phosphorylation, whereas SVST stimulates PP2A-dependent AKT phosphorylation. REF52 cells were infected with retroviruses containing either wild-type PyST or wild-type SVST or PyST or SVST mutated in either the PP2A or J domain or empty vector. Extracts of the infected cells were gel fractionated and probed with phospho-specific antibodies as (A) above. The amount of total ERK in each extract is shown. (C) PyST stimulates ERK phosphorylation and SVST stimulates AKT phosphorylation in both rat and human cells. Gel fractionated extracts from either rat REF52 cells or human BJ fibroblasts infected with retroviruses containing either no insert (empty) or PyST or SVST were probed with phospho-specific antibodies against ERK and AKT.

inhibit the ability of PyST to activate the MAP kinase pathway (Fig. 6A). However, mutation of the PP2A domain completely abrogates the ability of PyST expression to activate ERK (Fig. 6A). These data suggest that the cytoplasmic-localized MTA PyMT protein, but not the nuclear-localized MT11 protein, mimics PyST in activating the MAP kinase pathway by a mechanism dependent on the ability to bind to cellular PP2A. PyST and SVST Activate Different Cellular Signaling Pathways Involved in Growth Control. The small T-antigens of polyoma virus

(PyST) and SV40 (SVST) are similar in that both contain a J domain, which binds the Hsc70 heat shock protein, and a PP2A binding domain that allows it to mimic the cellular PP2A regulatory B subunit and bind to the core PP2A dimer composed of the A and C subunits (15, 16). SVST has been reported to activate the PI3-kinase pathway and activate AKT phosphorylation in human cells (17, 18). In addition, constitutive activation of PI3-kinase can substitute for the role of SVST in the transformation of hTERT immortalized normal human cells (18). We observed that PyST activated the MAP kinase pathway but not the PI3-kinase pathway in REF52 cells (Fig. 6A). Thus, we were interested in comparing the cellular pathways activated by PyST and SVST in REF52 cells. Fig. 6B shows the stimulation of ERK and AKT phosphorylation in REF52 cells after infection with retroviruses containing wild-type PyST, wild-type SVST, and mutants of each of the different small T-antigens in which either the J or PP2A binding domains are inactivated. PyST activated ERK, but not AKT, phosphorylation and this induction is dependent on a functional PP2A-binding domain. In clear contrast, SVST activated AKT, but not ERK, phosphorylation. Mutation of the PP2A-binding domain of SVST abrogates AKT phosphorylation, indicating that SVST activates the PI3K pathway in a manner dependent on PNAS 兩 December 19, 2006 兩 vol. 103 兩 no. 51 兩 19293

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PP2A binding. On the other hand, mutations in the J domain that inhibit binding to Hsc70 do not affect the induction of AKT phosphorylation by SVST or ERK phosphorylation by PyST (Fig. 6B). These results were not species specific because PyST activates the MAP kinase pathway and SVST activates AKT in human fibroblasts in a similar fashion as found in rat REF52 cells (Fig. 6C). Thus, our results indicate that the two different but related viral small T-antigens, through interaction with the same cellular PP2A AC dimer, can differentially modulate PP2A activity to regulate distinct signaling pathways critically involved in cell growth. Discussion Our original aim was to determine which cellular proteins are bound to the two previously isolated frameshift PyMT mutant proteins. Although we could show that PP2A A and C subunits were bound to both mutant PyMT proteins (Fig. 4), we found that neither mutant protein was associated with Src-family kinases or Shc proteins (Figs. 3 and 4). Thus, it was quite surprising to find that the cytoplasmic MTA protein activates the Raf-MEK-ERK MAP kinase cascade in the absence of Shc binding (Fig. 5). As the MTA PYMT protein contains 191 of the 195 amino acids present in PyST (Fig. 2), we reasoned that the MTA protein could be mimicking PyST in some of its properties. We found that PyST, in a similar fashion to the MTA protein, activated the MAP kinase pathway but did not stimulate the phosphorylation of AKT (Fig. 6). The activation of the MAP kinase pathway by both PyST and MTA proteins was dependent on the ability to act as a PP2A B subunit and bind to the PP2A dimer composed of the A and C subunits as inactivation of the PP2A domain of both these Py proteins inhibited the activation of the MAP kinase pathway (Figs. 5 and 6). PyST is reported to be located in both the cytoplasm and nucleus. MTA is located in the cytoplasm and stimulates ERK activity. On the other hand, MT11, which is located in the nucleus, is unable to activate ERK. This suggests that there may be a difference in the effect of the Py proteins on PP2A activity in different cellular locations. For instance, the Py proteins may replace different PP2A B subunits in the nucleus or the cytoplasm. Alternatively, the Py proteins may target their bound PP2A proteins to substrates that are only present at one of the two different cellular locations. We cannot rule out that cellular location is not playing a role and that the addition of the 85 PyLT and 6 vector amino acids at the C terminus of MT11 protein (Fig. 2) (8) is inhibiting its ability to activate the MAP kinase pathway. Both PyST and SVST are reported to bind to the PP2A AC dimer, but it was not clear until now that PyST and SVST may affect PP2A activity in different ways. The mechanism(s) used by the two ST proteins to modulate PP2A activity is also not clear. The different ST proteins may operate in a positive manner and act as a novel regulatory B subunit and direct the ST/PP2A A and C subunit complex to dephosphorylate a new target protein. The ST proteins may also act in a negative manner, replacing one or more B subunits in the PP2A holoenzyme and preventing the dephosphorylation of some of the B subunit-dependent target proteins. It is also possible that ST/PP2A complex acts both in a positive or negative fashion, affecting different target proteins. How could PP2A be involved in activating the MAP kinase pathway? One possibility is that the Py proteins positively modulate PP2A activity, leading to the activation of Raf, the most upstream kinase of the MAP kinase cascade. Raf kinases are subject to very complex regulatory processes that include stimulatory and inhibitory phosphorylation and dephosphorylation events (19, 20). PP2A is known to play a positive role in the activation of the MAP kinase pathway and it has been shown to interact with and dephosphorylate inhibitory sites in Raf-1 (21–24). PP2A also plays a positive role in MAP kinase pathway activation by dephosphorylation of Ksr, which is a scaffold-type 19294 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0609343103

protein that brings together the three kinases of the MAP kinase pathway and facilitates pathway activation (23). Another possibility is that PyST is acting negatively, and by replacing preferentially specific PP2A B subunits it is inhibiting the specific PP2A activities involved in the dephosphorylation of phospho Raf, phospho MEK, and phospho ERK. Inhibition of these specific PP2A activities would result in a change in the normal equilibrium between the phosphorylated and unphosphorylated forms of the MAP kinase proteins, resulting in an increase in the presence of the phosphorylated active forms that we detect. The PyST and SVST proteins have a similar structural organization. Both proteins contain a J domain near their amino terminus that can bind the Hsc70 heat shock protein and a PP2A binding domain that allows them to mimic the PP2A B subunit and bind to the PP2A A and C dimer. Our observation that the PyST/PP2A complex and the SVST/PP2A complex activate different cellular signaling pathways strongly indicates that the PyST and SVST proteins affect PP2A activity in different ways. The PP2A complexes containing each of the distinct ST proteins may target different cellular proteins involved in each of the dissimilar cellular pathways. Alternatively, PyST and SVST may replace different PP2A B subunits required for unique substrate specificity and the subsequent loss of these specific PP2A holoenzyme activities may differentially affect distinct cellular pathways. PyST is intimately associated with PyMT, being derived from the same genomic DNA, and the two proteins use the same and alternative reading frames as a result of alternative splicing. In a similar fashion, SVST is intimately associated with SV40 large T antigen (SVLT). PyST does not activate the PI3-kinase pathway, whereas PyMT can directly activate the PI3K pathway via its interaction with the PI3K p85 regulatory subunit. SVLT, on the other hand, does not activate the PI3-kinase pathway, but it is SVST that achieves PI3-kinase pathway activation. It may be that the activation of the cellular PI3-kinase pathway is essential for both the SV40 and Py life cycles, and the two viruses have evolved different mechanisms to activate this critical pathway. As PyMT stimulates the MAP kinase pathway via Ras activation (see Fig. 1) (1, 2), it is not clear why PyST should duplicate this activation. It could be that the PyST activation is related to differential spatiotemporal regulation of the pathway by the two Py proteins. It may also be that the major role of PyST is to target PP2A to regulate other distinct pathways important for Py life cycle and the activation of the MAP kinase pathway may be an indirect consequence of this effect of PyST on PP2A activity. Regardless, it is clear that the two related ST proteins from the polyoma and SV40 viruses, while binding to the same cellular PP2A AC dimer, have evolved to differentially modulate the activity of PP2A and target it to regulate different critical signaling pathways involved in cell growth. In summary, we have found that a cytoplasmic mutant of PyMT, as well as wild-type PyST can activate the Raf-MEK-ERK MAP kinase cascade. This activation appears to be independent of Ras and depends on the ability of these Py proteins to interact with PP2A. Whether this MAP kinase activation is the result of the PP2A complex containing the Py protein having a new specificity or that the presence of the Py protein in the PP2A complex results in the inhibition of specific PP2A activities is not clear at this time. Although both the PyST and the SVST each appear to bind to the PP2A AC dimer in a similar fashion, each of the ST proteins preferentially activate a different signaling pathway. Whereas PyST activates the Raf-MEK-ERK MAP kinase cascade, SVST activates the phosphorylation of AKT. This indicates that the effects of these ST proteins on PP2A activity are even more complex than originally envisioned. Rodriguez-Viciana et al.

Constructs and Retroviral Infections. PyMT, PyST, and SVST wild-type and mutant versions were cloned into the pENTRY vector (Invitrogen) and transferred to Gateway-compatible LXSN and LXSP3 retroviral vectors by using recombinationmediated Gateway technology (Invitrogen). Mutations were generated using the QuikChange site-directed mutagenesis kit (Stratagene). Retroviruses were generated by transient transfection of Phoenix packaging cells with Lipofectamine 2000 (Invitrogen). Polybrene was added to all retrovirus stocks to 5 ␮g/ml before REF52 cells were infected for 3–6 h at 37°C. Antibodies. The following antibodies were used: PYC for Western blot analysis with the polyoma T-Antigens (4). The PAb762 antibody used for PyMT immunoprecipitation was a gift from S. Dilworth (Imperial College, Hammersmith Hospital, London, U.K.). Raf-1 and Pan-Ras were from BD Transduction Laboratories. Phospho-T202/Y204 p44/42-ERK, total ERK, phosphoS338 Raf, and phospho-S259A Raf were from Cell Signalling. Phospho-S473 Akt was a gift from D. Stokoe (UCSF Cancer Research Institute). HRP-coupled secondary antibodies were from Amersham. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

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Western Blot Analysis and Immunoprecipitations. Cells were allowed to reach confluence and serum-starved overnight in 0.5% FBS. The next day, cells were washed using PBS and lysed in TNE lysis buffer: 20 mM Tris, pH 7.5/150 mM NaCl/1 mM EDTA/1% TX-100/1 mM DTT, containing protease and phosphatase inhibitor cocktails (Sigma). Where indicated, cleared lysates were immunoprecipitated with the PyMT or Raf-1 antibodies and protein G beads. After extensive washing, beads were resuspended in kinase buffer or in sample buffer. Protein lysates or immunoprecipitates were separated on 4–12% NuPAGE gels (Invitrogen). Proteins were transferred to PVDF membranes (Fisher), and membranes were incubated with the indicated antibodies. Bound proteins were detected with ECL (Amersham) or Visualizer (Upstate). Kinase Assays. PyMT proteins were immunoprecipitated from

cleared lysates with the PAb762 antibody and protein G beads. After four washes, associated kinase activity was assayed by performing a kinase assay. Briefly, beads were incubated for 30 min at room temperature in 20 mM Tris (pH 7.5)/50 mM NaCl/10 mM MgCl2/50 ␮M cold ATP/10 ␮Ci of [␣-32P]ATP (Amersham). Reactions were stopped with sample buffer and resolved by gel electrophoresis. Radioactive proteins were detected by autoradiography. We thank Drs. David Dankort, Gerard Evan, Frank McCormick, Martin McMahon, Clodagh O’Shea, and David Stokoe for their helpful advice and comments during the preparation of the manuscript and in the course of the research. This work was supported in part by National Institutes of Health Grants CA92454 and CA101967. 15. Brodsky JL, Pipas JM (1998) J Virol 72:5329–5334. 16. Moule MG, Collins CH, McCormick F, Fried M (2004) Proc Natl Acad Sci USA 101:14063–14066. 17. Yuan H, Veldman T, Rundell K, Schlegel R (2002) J Virol 76:10685–10691. 18. Zhao JJ, Gjoerup OV, Subramanian RR, Cheng Y, Chen W, Roberts TM, Hahn WC (2003) Cancer Cell 3:483–495. 19. Mercer KE, Pritchard CA (2003) Biochim Biophys Acta 1653:25–40. 20. Wellbrock C, Karasarides M, Marais R (2004) Nat Rev Mol Cell Biol 5:875–885. 21. Abraham D, Podar K, Pacher M, Kubicek M, Welzel N, Hemmings BA, Dilworth SM, Mischak H, Kolch W, Baccarini M (2000) J Biol Chem 275:22300–22304. 22. Dougherty MK, Muller J, Ritt DA, Zhou M, Zhou XZ, Copeland TD, Conrads TP, Veenstra TD, Lu KP, Morrison DK (2005) Mol Cell 17:215–224. 23. Ory S, Zhou M, Conrads TP, Veenstra TD, Morrison DK (2003) Curr Biol 13:1356–1364. 24. Sieburth DS, Sundaram M, Howard RM, Han M (1999) Genes Dev 13:2562– 2569. 25. Mor O, Read M, Fried M (1997) Oncogene 15:3113–3119.

PNAS 兩 December 19, 2006 兩 vol. 103 兩 no. 51 兩 19295

BIOCHEMISTRY

Materials and Methods Cell Culture. REF52 and DNp53REF52 cells have been described previously (4, 25). The DNp53REF52 cell line was established after infection of REF52 cells with pBabehygro-DNp53 (302– 390) (25). All cell lines were routinely cultured in Dulbecco’s modified Eagles medium supplemented with 10% FBS, 100 mg/ml penicillin, and 100 mg/ml streptomycin.