B55α PP2A Holoenzymes Modulate the Phosphorylation Status of the Retinoblastoma-related Protein p107 and Its Activation

Abstract

Pocket proteins negatively regulate transcription of E2F-dependent genes and progression through the G0/G1 transition and the cell cycle restriction point in G1. Pocket protein repressor activities are inactivated via phosphorylation at multiple Pro-directed Ser/Thr sites by the coordinated action of G1 and G1/S cyclin-dependent kinases. These phosphorylations are reversed by the action of two families of Ser/Thr phosphatases: PP1, which has been implicated in abrupt dephosphorylation of retinoblastoma protein (pRB) in mitosis, and PP2A, which plays a role in an equilibrium that counteracts cyclin-dependent kinase (CDK) action throughout the cell cycle. However, the identity of the trimeric PP2A holoenzyme(s) functioning in this process is unknown. Here we report the identification of a PP2A trimeric holoenzyme containing B55α, which plays a major role in restricting the phosphorylation state of p107 and inducing its activation in human cells. Our data also suggest targeted selectivity in the interaction of pocket proteins with distinct PP2A holoenzymes, which is likely necessary for simultaneous pocket protein activation. Pocket proteins negatively regulate transcription of E2F-dependent genes and progression through the G0/G1 transition and the cell cycle restriction point in G1. Pocket protein repressor activities are inactivated via phosphorylation at multiple Pro-directed Ser/Thr sites by the coordinated action of G1 and G1/S cyclin-dependent kinases. These phosphorylations are reversed by the action of two families of Ser/Thr phosphatases: PP1, which has been implicated in abrupt dephosphorylation of retinoblastoma protein (pRB) in mitosis, and PP2A, which plays a role in an equilibrium that counteracts cyclin-dependent kinase (CDK) action throughout the cell cycle. However, the identity of the trimeric PP2A holoenzyme(s) functioning in this process is unknown. Here we report the identification of a PP2A trimeric holoenzyme containing B55α, which plays a major role in restricting the phosphorylation state of p107 and inducing its activation in human cells. Our data also suggest targeted selectivity in the interaction of pocket proteins with distinct PP2A holoenzymes, which is likely necessary for simultaneous pocket protein activation. IntroductionThe retinoblastoma family of growth suppressor proteins, designated pocket proteins, includes the product of the retinoblastoma susceptibility gene and the functionally and structurally related proteins p107 and p130 (reviewed in Ref. 1Graña X. Garriga J. Mayol X. Oncogene. 1998; 17: 3365-3383Crossref PubMed Scopus (283) Google Scholar). Pocket proteins suppress the G0/G1 cell cycle transition and passage through the restriction point by repressing E2F-dependent transcription via a direct interaction of the hypophosphorylated pocket protein with members of the E2F family of transcription factors. Mitogen-dependent activation of D-type cyclin-CDK 2The abbreviations used are: CDKcyclin-dependent kinasepRBretinoblastoma proteinstsmall t antigen. complexes cooperates with cyclin E-CDK2 complexes to hyperphosphorylate pocket proteins starting in mid-to late G1 disrupting the pocket protein-E2F interaction and relieving repression of many genes whose products are required for cell cycle progression. Pocket proteins remain hyperphosphorylated through the S and G2 phases and part of mitosis as they become targets of cyclin-CDK complexes that operate at these cell cycle stages (reviewed in Refs. 2Sotillo E. Graña X. Enders G. Cell Cycle Deregulation in Cancer. Springer Publishing, New York2010: 3-22Crossref Google Scholar, 3Graña X. Cancer Biol. Ther. 2008; 7: 842-844Crossref PubMed Scopus (12) Google Scholar, 4Rowland B.D. Bernards R. Cell. 2006; 127: 871-874Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar).As cyclin-CDK complexes are inactivated during mitotic exit, the three pocket proteins become simultaneously hypophosphorylated. PP1 has been implicated in dephosphorylation of pRB from mitosis to a point in G1, where pRB becomes hyperphosphorylated by G1 cyclin-CDKs (reviewed in Refs. 3Graña X. Cancer Biol. Ther. 2008; 7: 842-844Crossref PubMed Scopus (12) Google Scholar and 5Tamrakar S. Rubin E. Ludlow J.W. Front Biosci. 2000; 5: D121-D137Crossref PubMed Google Scholar). However, a second phosphatase plays a role in reversing the action of CDKs throughout the cell cycle and in quiescent cells (6Garriga J. Jayaraman A.L. Limón A. Jayadeva G. Sotillo E. Truongcao M. Patsialou A. Wadzinski B.E. Graña X. Cell Cycle. 2004; 3: 1320-1330Crossref PubMed Scopus (45) Google Scholar). When the activity of cyclin-CDK complexes is pharmacologically inhibited, pocket proteins become rapidly dephosphorylated, and the severity of the hypophosphorylation depends on the subsets of CDKs that become inactivated. For instance, cycloheximide, an inhibitor of protein synthesis, causes rapid down-regulation of short lived cyclins such as D-type cyclins, resulting in potent dephosphorylation of p107, but only partial dephosphorylation of p130 and pRB, as CDK2 remains active for several hours. In contrast, treatment with the potent CDK inhibitor flavopiridol leads to rapid and complete dephosphorylation of pocket proteins as all CDKs become inactive (6Garriga J. Jayaraman A.L. Limón A. Jayadeva G. Sotillo E. Truongcao M. Patsialou A. Wadzinski B.E. Graña X. Cell Cycle. 2004; 3: 1320-1330Crossref PubMed Scopus (45) Google Scholar).PP2A, a second Ser/Thr phosphatase, has been implicated in dephosphorylation of pocket proteins throughout the cell cycle by using selective pharmacological inhibitors of PP2A in cells and in vitro, as well as by expressing in cells the Simian virus 40 (SV40) small t antigen (st), which binds and specifically inhibits PP2A (6Garriga J. Jayaraman A.L. Limón A. Jayadeva G. Sotillo E. Truongcao M. Patsialou A. Wadzinski B.E. Graña X. Cell Cycle. 2004; 3: 1320-1330Crossref PubMed Scopus (45) Google Scholar). In addition, the catalytic subunit of PP2A was found to associate with both p130 and p107 through the cell cycle and in quiescent cells. Others have implicated PP2A in dephosphorylation of pocket proteins in response to a variety of signals including oxidative stress (7Cicchillitti L. Fasanaro P. Biglioli P. Capogrossi M.C. Martelli F. J. Biol. Chem. 2003; 278: 19509-19517Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 8Magenta A. Fasanaro P. Romani S. Di Stefano V. Capogrossi M.C. Martelli F. Mol. Cell. Biol. 2008; 28: 873-882Crossref PubMed Scopus (50) Google Scholar), DNA damage (9Avni D. Yang H. Martelli F. Hofmann F. ElShamy W.M. Ganesan S. Scully R. Livingston D.M. Mol. Cell. 2003; 12: 735-746Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), retinoic acid (10Vuocolo S. Purev E. Zhang D. Bartek J. Hansen K. Soprano D.R. Soprano K.J. J. Biol. Chem. 2003; 278: 41881-41889Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), UV radiation (11Voorhoeve P.M. Watson R.J. Farlie P.G. Bernards R. Lam E.W. Oncogene. PubMed Scopus Google Scholar), and in cells V. E. 2008; PubMed Scopus Google cells a of distinct protein many of that in the of proteins with catalytic phosphatase activity is many of the also has been that a role with in the of protein However, has that PP2A, its selectivity that is by with multiple (reviewed in Ref. D.M. S. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). PP2A is a trimeric that of a catalytic subunit a subunit and a subunit genes for and of these However, are families of and with several members by genes with multiple that and proteins M. A. S. F. R. Mol. Cell Full Text Full Text PDF PubMed Scopus Google Scholar). that in of distinct PP2A with distinct (reviewed in Ref. D.M. S. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google we implicated PP2A in the phosphorylation state of pocket proteins as part of a equilibrium with CDKs throughout the cell the holoenzyme pocket proteins in this is unknown. have used in and to as a subunit that a PP2A trimeric that targets p107 and trimeric holoenzyme specifically p107 in of in cells in the phosphorylation of p107 to a that the of this subunit the phosphorylation state of these pocket Our data also that PP2A pocket proteins as pRB to associate with a small in is to p107 but to growth as the pocket proteins remain that multiple distinct PP2A pocket proteins to have that PP2A plays a direct role in restricting the phosphorylation of pocket proteins through the cell cycle (6Garriga J. Jayaraman A.L. Limón A. Jayadeva G. Sotillo E. Truongcao M. Patsialou A. Wadzinski B.E. Graña X. Cell Cycle. 2004; 3: 1320-1330Crossref PubMed Scopus (45) Google Scholar). we have p130 and p107 with the catalytic subunit of PP2A and implicated an holoenzyme in phosphorylation of pocket proteins, the of the is pocket proteins complexes with of the PP2A we pocket proteins and from of human cells. of an of on with cell of cells and by and of and was by with the that and p130 was of the of this was using of an of of that by for to and but we of PP2A to the of was that of the of also this interaction in of human cells that been and with for of that that is complexes with PP2A in the of quiescent as well as cells at of the cell cycle. is in with that with through the cell cycle (6Garriga J. Jayaraman A.L. Limón A. Jayadeva G. Sotillo E. Truongcao M. Patsialou A. Wadzinski B.E. Graña X. Cell Cycle. 2004; 3: 1320-1330Crossref PubMed Scopus (45) Google Scholar).As the of with both and that p107 was PP2A holoenzymes, we to p107 a for with B55α, and which are members of the and of trimeric complexes that in subunit in cells. was with which complexes as complexes by the of as we an of PP2A in and was by by that trimeric complexes containing the subunit by of both and was that the by complexes containing the of subunit from as with the was the to of the trimeric However, the the of trimeric was and was the selectivity of p107 for the subunit with the of and that the subunit is the PP2A to p107 and that is the this in selectivity in we cells with the in and from cell by was only was but was only with was with in with PP2A in the of p107 with also complexes in cells. and was to a which from the of p107 that These data of which to the of the an interaction p107 and PP2A both in and in we pocket proteins also with that and but with pocket proteins are with the and at was also in in using of cells with the in with and to a with with both and at with but this that pocket proteins are targeted by distinct PP2A with to at two of distinct subunit family pocket with p107 and p130 but pRB in cells expressing was as in is by an cells with at with of the and via are by a in the for have as are in the pocket proteins that are related in in pRB, and these pocket we a of p107 to the in p107 that the interaction with the PP2A that well on p107 via and are in using these that the of p107 is for to and as well the cyclin which is to with the of In contrast, a in the of the In multiple we also that the was to a with PP2A the to the and the of the in the of an protein is the using trimeric that the of p107 but the with B55α, but the is to the the interaction of this is with that of the with of p107 the interaction with the and the the p107 and activity with PP2A are the of p107 is to with and and the of p107 this interaction but to with with of and proteins with of cells. by but with the of with the proteins and as in of that the of a and a p107 both with the acid that the three and the acid that the in in cells However, these of with and and has been that a of B55α, also with both Yang J. X. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These suggest that multiple with the also a of point in the of the that have been found to for dephosphorylation of by Zhang P. Mol. Cell. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). that only was for to p107 in from of cells with the was also for of to and is for both of an holoenzyme and p107 also that of to p107 to and and required for to of and in its that are for dephosphorylation of the is for the of cells with and the Cell with and and via using and of p107 for trimeric containing and the of p107 to that and that the we have that of in cells dephosphorylation of pocket proteins CDK activity is (6Garriga J. Jayaraman A.L. Limón A. Jayadeva G. Sotillo E. Truongcao M. Patsialou A. Wadzinski B.E. Graña X. Cell Cycle. 2004; 3: 1320-1330Crossref PubMed Scopus (45) Google Scholar), that of trimeric PP2A complexes with cells with B55α, and and with expressing of the of but the of the specifically with and but with of the interaction of and with but with the and using cells with B55α, and we an interaction and of trimeric complexes by of the subunit from in the interaction and but and with p107 of the of the PP2A of PP2A by the of in dephosphorylation of pocket proteins the activity of CDKs is (6Garriga J. Jayaraman A.L. Limón A. Jayadeva G. Sotillo E. Truongcao M. Patsialou A. Wadzinski B.E. Graña X. Cell Cycle. 2004; 3: 1320-1330Crossref PubMed Scopus (45) Google and an interaction the and and antigen t in cells of PP2A and but to and by cells with and and with and complexes by by with the of on PP2A complexes are of cells as in with and the of with in the of was via interaction the holoenzyme and p107 and interaction p107 is a of this p107 a of PP2A p107 is a of the p107 was in with cyclin complexes and and with complexes in products by and by dephosphorylation of CDK the dephosphorylation is we to the activity of and this we the of holoenzyme required to dephosphorylation of a that by both that at but In contrast, both These that the subunit of the holoenzyme is for of p107 as a in trimeric PP2A complexes p107 in of complexes with in with cyclin of and with with cyclin p107 is a of in vitro, of its activity in cells the phosphorylation of this is the we the of expressing that of at with in a in the phosphorylation of p107, its is also for which only in the in a on p107 of the of that p107 in the cell also that these dephosphorylation of leads to a on p107 which was by of leads to potent of p107, that PP2A are required to the phosphorylation state that is from cells. but dephosphorylation of p130 was and to a in the of which from and of the hyperphosphorylated of p130 D. J. PubMed Scopus Google Scholar, S. Garriga J. J. Graña X. Oncogene. 2003; PubMed Scopus Google Scholar). we the of and using a that of leads to an in that is its in cells. these data suggest that the of in the cells are to the phosphorylation state of of B55α, but in dephosphorylation of p107 and cells with the at and on the phosphorylation of p107 and p130 was via and hypophosphorylated are by and in leads to p107 cells as in with the of was as in A. was with cells as in and with in phosphorylation by that leads to potent of of and with of was with p107 and hypophosphorylated p130 are by an the of are to the phosphorylation state of p107, of in p107 cells with used as that of with the of was with which also in the by and the of and that are to of of B55α, but in of cells with the to B55α, and p107 phosphorylation and via and hypophosphorylated are by and is for the we cells with B55α, but p107 dephosphorylation These that dephosphorylation of p107 is on the interaction the of the these cells dephosphorylation of p107, that selective dephosphorylation of p107 in cells is for growth likely p130 and pRB remain at hyperphosphorylated and to in repressing with this the of cyclin and p107, all gene are with of dephosphorylation of p107 leads to of that p107 is but to E2F-dependent gene in the of activation of p130 and In with this cell cycle by in cells of but a in p107 p107 activation. of but a p107 dephosphorylation the of E2F-dependent gene cells with expressing B55α, and protein by with that in this p130 have been to the dephosphorylation and and hypophosphorylated are by and of protein with and complexes by by with to p107 and these suggest that is a of a that of pocket proteins with a for distinct PP2A are required in to to simultaneously CDK action the three pocket was implicated as the phosphatase that pocket proteins to an active state the activity of CDKs is (6Garriga J. Jayaraman A.L. Limón A. Jayadeva G. Sotillo E. Truongcao M. Patsialou A. Wadzinski B.E. Graña X. Cell Cycle. 2004; 3: 1320-1330Crossref PubMed Scopus (45) Google Scholar). However, PP2A is a family of trimeric containing of many that the PP2A that pocket proteins as part of the equilibrium with CDKs that the phosphorylation state of pocket proteins throughout the cell cycle are Here we have a trimeric that the catalytic and of PP2A and B55α, a of the trimeric specifically with p107 and p130 in and in cells. interaction is direct and by the which targets the of Our data that the interaction with is to as pRB Our also that the of are to the of p107 phosphorylation that are in these suggest that an active role in the phosphorylation of p107 and its activation state the dephosphorylation of p107, and to a the activity of CDKs is with the of activation of both p107 and p130 for growth by CDK inhibitors M. J. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), the selective activation of p107 by is to growth and as is the with multiple PP2A are required to simultaneously pocket have implicated PP2A as a of pocket protein phosphorylation and role of PP2A in pocket protein by two of that is in and that is In of the PP2A and phosphorylation of pocket proteins in throughout the cell cycle and in quiescent cells (6Garriga J. Jayaraman A.L. Limón A. Jayadeva G. Sotillo E. Truongcao M. Patsialou A. Wadzinski B.E. Graña X. Cell Cycle. 2004; 3: 1320-1330Crossref PubMed Scopus (45) Google Scholar). for the PP2A is in response to a variety of signals to pocket proteins (7Cicchillitti L. Fasanaro P. Biglioli P. Capogrossi M.C. Martelli F. J. Biol. Chem. 2003; 278: 19509-19517Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 8Magenta A. Fasanaro P. Romani S. Di Stefano V. Capogrossi M.C. Martelli F. Mol. Cell. Biol. 2008; 28: 873-882Crossref PubMed Scopus (50) Google Scholar, D. Yang H. Martelli F. Hofmann F. ElShamy W.M. Ganesan S. Scully R. Livingston D.M. Mol. Cell. 2003; 12: 735-746Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, S. Purev E. Zhang D. Bartek J. Hansen K. Soprano D.R. Soprano K.J. J. Biol. Chem. 2003; 278: 41881-41889Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, P.M. Watson R.J. Farlie P.G. Bernards R. Lam E.W. Oncogene. PubMed Scopus Google Scholar, V. E. 2008; PubMed Scopus Google Scholar). that PP2A to pocket proteins in response to a variety of signals are to stress a subunit from the to associate with pRB its dephosphorylation (7Cicchillitti L. Fasanaro P. Biglioli P. Capogrossi M.C. Martelli F. J. Biol. Chem. 2003; 278: 19509-19517Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 8Magenta A. Fasanaro P. Romani S. Di Stefano V. Capogrossi M.C. Martelli F. Mol. Cell. Biol. 2008; 28: 873-882Crossref PubMed Scopus (50) Google Scholar). has that rapid dephosphorylation of p107 in response to in a cell is by a PP2A containing a subunit from the the family as dephosphorylation is to an protein that inhibits these V. E. 2008; PubMed Scopus Google Scholar). However, the PP2A to pocket proteins in response to signals remain to have that PP2A phosphorylation of pocket proteins the activity of CDKs is (6Garriga J. Jayaraman A.L. Limón A. Jayadeva G. Sotillo E. Truongcao M. Patsialou A. Wadzinski B.E. Graña X. Cell Cycle. 2004; 3: 1320-1330Crossref PubMed Scopus (45) Google Scholar). In this we that of in dephosphorylation of p107, in of p107 in cells expressing of is on to a with These data that the holoenzyme is in cells at that of pocket of cells with in of p107 to in cells at point of the cell cycle is a potent inhibitor of the targets PP2A in to complexes that to pocket protein are complexes as we have that they are also to is that of and complexes to phosphorylation of pocket proteins in the of that to activation of PP2A complexes including of PP2A complexes in to in an equilibrium with CDKs is from and data as CDK in coordinated dephosphorylation of the three pocket proteins (6Garriga J. Jayaraman A.L. Limón A. Jayadeva G. Sotillo E. Truongcao M. Patsialou A. Wadzinski B.E. Graña X. Cell Cycle. 2004; 3: 1320-1330Crossref PubMed Scopus (45) Google Scholar), but complexes p107 and p130 pRB we have found that with the of p107 that is on but in pRB, is a for the selectivity of these In contrast, with both the and the pocket of pRB A. Fasanaro P. Romani S. Di Stefano V. Capogrossi M.C. Martelli F. Mol. Cell. Biol. 2008; 28: 873-882Crossref PubMed Scopus (50) Google Scholar), a of interaction that with the three pocket proteins with remains to both PP2A CDK sites they for distinct subsets of of a phosphatase PP1 in the activation of pocket proteins has been in where PP1 is required for of and the of genes in where pRB is that phosphatase with PP1 is for pRB activation R.J. PubMed Scopus (12) Google is the of in the phosphorylation of p107 and p130 in cells. in p107 by is as leads to complete of active p107 which are for restricting E2F-dependent in p130 is also which from phosphorylation of the CDK sites that p130 and by the D. J. PubMed Scopus Google Scholar, S. Garriga J. J. Graña X. Oncogene. 2003; PubMed Scopus Google Scholar). is that the of on pocket protein phosphorylation of pocket proteins, to of cell by However, the of in which CDK are in with the of in quiescent where to the phosphorylation of pocket proteins, likely as a of the of CDK activity E. Garriga J. A. Graña X. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). the of p107 phosphorylation to the of B55α, of pocket proteins is to of that direct PP2A to pocket IntroductionThe retinoblastoma family of growth suppressor proteins, designated pocket proteins, includes the product of the retinoblastoma susceptibility gene and the functionally and structurally related proteins p107 and p130 (reviewed in Ref. 1Graña X. Garriga J. Mayol X. Oncogene. 1998; 17: 3365-3383Crossref PubMed Scopus (283) Google Scholar). Pocket proteins suppress the G0/G1 cell cycle transition and passage through the restriction point by repressing E2F-dependent transcription via a direct interaction of the hypophosphorylated pocket protein with members of the E2F family of transcription factors. Mitogen-dependent activation of D-type cyclin-CDK 2The abbreviations used are: CDKcyclin-dependent kinasepRBretinoblastoma proteinstsmall t antigen. complexes cooperates with cyclin E-CDK2 complexes to hyperphosphorylate pocket proteins starting in mid-to late G1 disrupting the pocket protein-E2F interaction and relieving repression of many genes whose products are required for cell cycle progression. Pocket proteins remain hyperphosphorylated through the S and G2 phases and part of mitosis as they become targets of cyclin-CDK complexes that operate at these cell cycle stages (reviewed in Refs. 2Sotillo E. Graña X. Enders G. Cell Cycle Deregulation in Cancer. Springer Publishing, New York2010: 3-22Crossref Google Scholar, 3Graña X. Cancer Biol. Ther. 2008; 7: 842-844Crossref PubMed Scopus (12) Google Scholar, 4Rowland B.D. Bernards R. Cell. 2006; 127: 871-874Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar).As cyclin-CDK complexes are inactivated during mitotic exit, the three pocket proteins become simultaneously hypophosphorylated. PP1 has been implicated in dephosphorylation of pRB from mitosis to a point in G1, where pRB becomes hyperphosphorylated by G1 cyclin-CDKs (reviewed in Refs. 3Graña X. Cancer Biol. Ther. 2008; 7: 842-844Crossref PubMed Scopus (12) Google Scholar and 5Tamrakar S. Rubin E. Ludlow J.W. Front Biosci. 2000; 5: D121-D137Crossref PubMed Google Scholar). However, a second phosphatase plays a role in reversing the action of CDKs throughout the cell cycle and in quiescent cells (6Garriga J. Jayaraman A.L. Limón A. Jayadeva G. Sotillo E. Truongcao M. Patsialou A. Wadzinski B.E. Graña X. Cell Cycle. 2004; 3: 1320-1330Crossref PubMed Scopus (45) Google Scholar). When the activity of cyclin-CDK complexes is pharmacologically inhibited, pocket proteins become rapidly dephosphorylated, and the severity of the hypophosphorylation depends on the subsets of CDKs that become inactivated. For instance, cycloheximide, an inhibitor of protein synthesis, causes rapid down-regulation of short lived cyclins such as D-type cyclins, resulting in potent dephosphorylation of p107, but only partial dephosphorylation of p130 and pRB, as CDK2 remains active for several hours. In contrast, treatment with the potent CDK inhibitor flavopiridol leads to rapid and complete dephosphorylation of pocket proteins as all CDKs become inactive (6Garriga J. Jayaraman A.L. Limón A. Jayadeva G. Sotillo E. Truongcao M. Patsialou A. Wadzinski B.E. Graña X. Cell Cycle. 2004; 3: 1320-1330Crossref PubMed Scopus (45) Google Scholar).PP2A, a second Ser/Thr phosphatase, has been implicated in dephosphorylation of pocket proteins throughout the cell cycle by using selective pharmacological inhibitors of PP2A in cells and in vitro, as well as by expressing in cells the Simian virus 40 (SV40) small t antigen (st), which binds and specifically inhibits PP2A (6Garriga J. Jayaraman A.L. Limón A. Jayadeva G. Sotillo E. Truongcao M. Patsialou A. Wadzinski B.E. Graña X. Cell Cycle. 2004; 3: 1320-1330Crossref PubMed Scopus (45) Google Scholar). In addition, the catalytic subunit of PP2A was found to associate with both p130 and p107 through the cell cycle and in quiescent cells. Others have implicated PP2A in dephosphorylation of pocket proteins in response to a variety of signals including oxidative stress (7Cicchillitti L. Fasanaro P. Biglioli P. Capogrossi M.C. Martelli F. J. Biol. Chem. 2003; 278: 19509-19517Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 8Magenta A. Fasanaro P. Romani S. Di Stefano V. Capogrossi M.C. Martelli F. Mol. Cell. Biol. 2008; 28: 873-882Crossref PubMed Scopus (50) Google Scholar), DNA damage (9Avni D. Yang H. Martelli F. Hofmann F. ElShamy W.M. Ganesan S. Scully R. Livingston D.M. Mol. Cell. 2003; 12: 735-746Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), retinoic acid (10Vuocolo S. Purev E. Zhang D. Bartek J. Hansen K. Soprano D.R. Soprano K.J. J. Biol. Chem. 2003; 278: 41881-41889Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), UV radiation (11Voorhoeve P.M. Watson R.J. Farlie P.G. Bernards R. Lam E.W. Oncogene. PubMed Scopus Google Scholar), and in cells V. E. 2008; PubMed Scopus Google cells a of distinct protein many of that in the of proteins with catalytic phosphatase activity is many of the also has been that a role with in the of protein However, has that PP2A, its selectivity that is by with multiple (reviewed in Ref. D.M. S. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). PP2A is a trimeric that of a catalytic subunit a subunit and a subunit genes for and of these However, are families of and with several members by genes with multiple that and proteins M. A. S. F. R. Mol. Cell Full Text Full Text PDF PubMed Scopus Google Scholar). that in of distinct PP2A with distinct (reviewed in Ref. D.M. S. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google we implicated PP2A in the phosphorylation state of pocket proteins as part of a equilibrium with CDKs throughout the cell the holoenzyme pocket proteins in this is unknown. have used in and to as a subunit that a PP2A trimeric that targets p107 and trimeric holoenzyme specifically p107 in of in cells in the phosphorylation of p107 to a that the of this subunit the phosphorylation state of these pocket Our data also that PP2A pocket proteins as pRB to associate with a small in is to p107 but to growth as the pocket proteins remain that multiple distinct PP2A pocket proteins to activation.