Distinct Roles for PP1 and PP2A in Phosphorylation of the Retinoblastoma Protein

The function of the retinoblastoma protein (pRB) in controlling the G1 to S transition is regulated by phosphorylation and dephosphorylation on serine and threonine residues. While the roles of cyclin-dependent kinases in phosphorylating and inactivating pRB have been characterized in detail, the roles of protein phosphatases in regulating the G1/S transition are not as well understood. We used cell-permeable inhibitors of protein phosphatases 1 and 2A to assess the contributions of these phosphatases in regulating cyclin-dependent kinase activity and pRB phosphorylation. Treating asynchronously growing Balb/c 3T3 cells with PP2A-selective concentrations of either okadaic acid or calyculin A caused a time- and dose-dependent decrease in pRB phosphorylation. Okadaic acid and calyculin A had no effect on pRB phosphatase activity even though PP2A was completely inhibited. The decrease in pRB phosphorylation correlated with inhibitor-induced suppression of G1cyclin-dependent kinases including CDK2, CDK4, and CDK6. The inhibitors also caused decreases in the levels of cyclin D2 and cyclin E, and induction of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1. The decrease in cyclin-dependent kinase activities were not dependent on induction of cyclin-dependent kinase inhibitors since CDK inhibition still occurred in the presence of actinomycin D or cycloheximide. In contrast, selective inhibition of protein phosphatase 1 with tautomycin inhibited pRB phosphatase activity and maintained pRB in a highly phosphorylated state. The results show that protein phosphatase 1 and protein phosphatase 2A, or 2A-like phosphatases, play distinct roles in regulating pRB function. Protein phosphatase 1 is associated with the direct dephosphorylation of pRB while protein phosphatase 2A is involved in pathways regulating G1cyclin-dependent kinase activity. The function of the retinoblastoma protein (pRB) in controlling the G1 to S transition is regulated by phosphorylation and dephosphorylation on serine and threonine residues. While the roles of cyclin-dependent kinases in phosphorylating and inactivating pRB have been characterized in detail, the roles of protein phosphatases in regulating the G1/S transition are not as well understood. We used cell-permeable inhibitors of protein phosphatases 1 and 2A to assess the contributions of these phosphatases in regulating cyclin-dependent kinase activity and pRB phosphorylation. Treating asynchronously growing Balb/c 3T3 cells with PP2A-selective concentrations of either okadaic acid or calyculin A caused a time- and dose-dependent decrease in pRB phosphorylation. Okadaic acid and calyculin A had no effect on pRB phosphatase activity even though PP2A was completely inhibited. The decrease in pRB phosphorylation correlated with inhibitor-induced suppression of G1cyclin-dependent kinases including CDK2, CDK4, and CDK6. The inhibitors also caused decreases in the levels of cyclin D2 and cyclin E, and induction of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1. The decrease in cyclin-dependent kinase activities were not dependent on induction of cyclin-dependent kinase inhibitors since CDK inhibition still occurred in the presence of actinomycin D or cycloheximide. In contrast, selective inhibition of protein phosphatase 1 with tautomycin inhibited pRB phosphatase activity and maintained pRB in a highly phosphorylated state. The results show that protein phosphatase 1 and protein phosphatase 2A, or 2A-like phosphatases, play distinct roles in regulating pRB function. Protein phosphatase 1 is associated with the direct dephosphorylation of pRB while protein phosphatase 2A is involved in pathways regulating G1cyclin-dependent kinase activity. retinoblastoma protein cyclin-dependent kinase protein serine-threonine phosphatase 1 protein serine-threonine phosphatase 2A okadaic acid calyculin A tautomycin dithiothreitol glutathione S-transferase The retinoblastoma tumor suppressor gene encodes a nuclear phosphoprotein (pRB)1 that regulates the G1/S transition of the cell cycle. The active form of pRB binds and inactivates transcription factors, including members of the E2F family, whose target genes are necessary for S phase (1Riley D.J. Lee E.Y. Lee W.H. Annu. Rev. Cell Biol. 1994; 10: 1-29Crossref PubMed Scopus (203) Google Scholar, 2Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4347) Google Scholar, 3Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (5016) Google Scholar). The activity of pRB is regulated by phosphorylation and dephosphorylation of serine and threonine residues. pRB is dephosphorylated during mitosis, and the active, hypophosphorylated form inhibits cell cycle progression during early and mid G1 (4Ludlow J.W. Shon J. Pipas J.M. Livingston D.M. DeCaprio J.A. Cell. 1990; 60: 387-396Abstract Full Text PDF PubMed Scopus (294) Google Scholar). pRB accumulates in the inactive, hyperphosphorylated state in late G1 and phosphorylation is maintained during S and G2. Hyperphosphorylation causes dissociation of pRB from E2F, induction of gene transcription, and progression into S phase (5Chellappan S.P. Hiebert S. Mudryj M. Horowitz J.M. Nevins J.R. Cell. 1991; 65: 1053-1061Abstract Full Text PDF PubMed Scopus (1107) Google Scholar). Because of its central role in progression through G1, pRB serves as an important point of integration for numerous signaling pathways that influence the cell cycle (2Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4347) Google Scholar). pRB is phosphorylated by members of the cyclin-dependent family of serine/threonine kinases whose active forms consist of a catalytic subunit (CDK) complexed with a cyclin partner (2Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4347) Google Scholar, 3Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (5016) Google Scholar). The major kinases that phosphorylate pRB during G1 include cyclin E-CDK2, cyclin D-CDK4, and cyclin D-CDK6. pRB phosphorylation is initiated in a growth factor-dependent manner by assembly of cyclin D with CDK4. Phosphorylation is then accelerated during late G1 by cyclin E-CDK2 (6Matsuoka M. Kato J.Y. Fisher R.P. Morgan D.O. Sherr C.J. Mol. Cell. Biol. 1994; 14: 7265-7275Crossref PubMed Scopus (186) Google Scholar). Maintenance of the phosphorylated state during S and G2 is due to the actions of cyclin A- and cyclin B-CDK complexes. Cyclin-CDK complexes phosphorylate multiple proline-directed consensus sites on pRB (7Lees J.A. Buchkovich K.J. Marshak D.R. Anderson C.W. Harlow E. EMBO J. 1991; 10: 4279-4290Crossref PubMed Scopus (269) Google Scholar). The activities of G1 CDKs are regulated not only by the availability of cyclins, but also by activating (8Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2951) Google Scholar) and inhibitory M. S. Nature. 1995; PubMed Scopus Google Scholar, J. Nature. PubMed Scopus Google Scholar) phosphorylation of the CDK catalytic CDK activity is by cyclin-dependent kinase of G1 CDKs include and are also inhibited by a of CDK inhibitors J.W. Cell Biol. 1994; PubMed Scopus Google Scholar, C.J. J.M. 1995; PubMed Scopus Google Scholar, 1996; Scholar). pRB is by dephosphorylation the of Protein phosphatase 1 been as the major pRB phosphatase in of pRB by is to play a role in controlling the G1/S transition J.W. Biol. 1995; PubMed Scopus Google Scholar). 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PP2A and phosphatases are to okadaic acid these results that the of in G1 due to suppression of also that PP2A activity is for into S The dephosphorylation of pRB in to okadaic acid is and that an okadaic phosphatase While these results that protein phosphatases are involved in the of the G1/S transition and the of pRB the sites of of are not are highly inhibitors of members of the family of serine/threonine phosphatases 1994; Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). The to these inhibitors to and the levels of and PP2A in cell and S. PubMed Scopus Google Scholar). of these inhibitors including okadaic calyculin and tautomycin are and phosphatase activity in to for and PP2A and distinct these inhibitors and PP2A in a highly selective manner J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). We used these inhibitors to the roles and of and PP2A in regulating pRB phosphorylation. The results that phosphatases are involved in controlling the of pRB phosphorylation but that through distinct or a is for or of G1 cyclin-dependent kinase while Balb/c 3T3 cells were maintained in in an of cells cells were in calyculin A okadaic acid or tautomycin and were by in and was in the of and and an the phosphorylated and forms of pRB from a of and were The cyclin D was a that with and from a of cyclin cyclin D2 and cyclin were that the cyclin a and a tumor was used as a J.W. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). is a that with the of a and a is a that The pRB protein used as in the kinase and phosphatase was a glutathione S-transferase with the of pRB cells with and were as J.W. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) that the for CDK was 1 1 1 and CDKs were for kinase activity or by and by with The phosphorylation state of pRB was by the of the and hypophosphorylated forms during J.W. DeCaprio J.A. Lee E. Livingston D.M. Cell. Full Text PDF PubMed Scopus Google Scholar). Balb/c 3T3 cells were with the and in the were and of of protein were with The complexes were with protein with and and with kinase 1 1 and 1 complexes were with 1 of and of for The kinase were by of E. A Scholar) and for The were by on a and to or with a and were with as J. Biol. Full Text PDF PubMed Google Scholar). pRB was phosphorylated with the cyclin from of 1 Balb/c 3T3 cells with was in kinase with of and of The kinase was for with was by for and the to a Protein to The had been with and 1 Cell used for phosphatase were a of the by and S. PubMed Scopus Google Scholar). Balb/c 3T3 cells were for with or or for with the concentrations in the were by and with cell were for on in of 1 1 and The was through a to was by for The was through a with 1 1 and to that with the protein phosphatase The cell were and was only for phosphatase The activities of protein phosphatases 1 and 2A in cell were by the of or J. Biol. Full Text PDF PubMed Google Scholar). The activities of and PP2A were by to 1991; PubMed Scopus Google Scholar). activity was as the activity that was to Protein phosphatase 2A, and were as the activity that was inhibited by a protein phosphatase activity was also in the presence of 1 completely inhibits and the cell were to the of for a of the is necessary in to and PP2A levels 1991; PubMed Scopus Google Scholar). were the of with of the and dependent on the of phosphatase activity was as J.W. Livingston D.M. DeCaprio J.A. Mol. Cell. Biol. PubMed Scopus Google Scholar). were in a of in phosphatase 1 with 1 of and of cell The were by of and for The were by on The were and to or or 1 was in the was with the cell for on to the of Okadaic calyculin and tautomycin and PP2A in and cell J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The of these inhibitors in the contributions of and PP2A to the of pRB phosphorylation was by on and PP2A activity in Balb/c 3T3 cells were with the inhibitors and the activities of and PP2A were in cell were with a for and PP2A S. PubMed Scopus Google and with a highly selective for PP2A E. J. Cell Biol. 1995; PubMed Scopus Google Scholar). that inhibition of PP2A in Balb/c 3T3 cells 1 and The of for inhibition of PP2A was to and was due to the for to J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). of Balb/c 3T3 cells with or caused highly selective inhibition of growing Balb/c 3T3 cells were with 1 or for were and for phosphatase activity and as characterized were used to the activities of and PP2A in the cell S. PubMed Scopus Google Scholar). phosphatase were in the of in the presence of to or in the presence of 1 to completely PP2A and these the of phosphatase activity to PP2A to the activity inhibited by while the to to the activity to but inhibited by 1 of to of cells inhibited activity by 1 that for the of the activity in Balb/c 3T3 of 1 in inhibition of phosphatase activity. and are not inhibited by 1 S. PubMed Scopus Google that and PP2A were the major activities in Balb/c 3T3 In contrast, of caused inhibition of phosphatase activity that PP2A and for of the activity 1 of Balb/c 3T3 cells with either or caused a in phosphatase activity in the of 1 of to from cells had no effect that the phosphatase activity was due to The that the activity in from cells was to the activity in from cells that and inhibited of the PP2A activity in Balb/c 3T3 cells but had on The activity in from or cells were the activity from cells but the were not The of and to inhibition of PP2A was in activity was completely by with either 1 from these were that of Balb/c 3T3 cells with 1 or caused inhibition of PP2A activity and effect on activity. and have for a for PP2A M. J. 1995; PubMed Scopus Google Scholar) and inhibits in cells J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). on that inhibition of a of and Balb/c 3T3 cells were with for of from cells that in a decrease in activity and a of was activity that was to 1 was also a decrease in PP2A since the phosphatase activity that was to was also A decrease in PP2A activity was also as the 1 The PP2A activity from cells was to of that from that of Balb/c 3T3 cells with in inhibition of activity and a decrease in PP2A activity. The cell were to phosphatase to the of for of was necessary in to and PP2A activity S. PubMed Scopus Google Scholar). was that the inhibitors to and PP2A of cells cell or were the the activities from cells to from cells with of the was by and PP2A activities of the with and The phosphatase activities in from and cells not the were from to not results that was dissociation of the inhibitors from either or PP2A during and to The of dissociation of and from and PP2A was with the of these M. 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The of and on phosphorylation of the retinoblastoma protein were in Balb/c 3T3 cells growing asynchronously in of cells were with and in pRB phosphorylation by during pRB was in the hyperphosphorylated state in cells or in cells with The of the major protein as the and forms of pRB were by the with The was a protein since was also in with the 1 results were pRB was with a of cells with concentrations of caused dephosphorylation of pRB also caused dephosphorylation of pRB but the concentrations of were for dephosphorylation was and dephosphorylation was In dephosphorylation of pRB with the concentrations for inhibition of to an in the of pRB in the The of the in the of pRB is not We not an in pRB by of from cells even though pRB was completely dephosphorylated In to and had no effect on pRB phosphorylation pRB was maintained in the highly phosphorylated form of Balb/c 3T3 cells with a that caused inhibition of dephosphorylation of In dephosphorylation was 1 of pRB was not dependent on or protein since with either actinomycin D or had no effect on dephosphorylation of pRB not of CDK Balb/c 3T3 cells were with 1 and actinomycin D and or and for 1 was then to of cells and and from the and for a were as or as were on and by Balb/c 3T3 cells were with actinomycin D or 1 and for 1 was to of cells for an The of or in cell were by that pRB is dephosphorylated by a of Balb/c 3T3 cells with or caused dephosphorylation of pRB while maintained the hyperphosphorylated form of that the dephosphorylation of pRB by and was due to direct dephosphorylation of and from the selective inhibition of PP2A by these were the a pRB phosphatase active cells were with and but not with was by phosphatase activity in cell pRB as the from Balb/c 3T3 cells with or 1 were with pRB and phosphatase activity by from cells with dephosphorylated pRB The pRB phosphatase activity was due to since was to but was by 1 and Treating Balb/c 3T3 cells with either or for had no effect on pRB phosphatase activity. pRB was dephosphorylated by from either or In the pRB phosphatase activity to since was to and to 1 and and In contrast, was a in pRB phosphatase activity in of cells with The pRB phosphatase activity was to and inhibited by 1 was the of pRB phosphorylation dephosphorylation by the had to A in pRB phosphatase activity in from cells was also dephosphorylation was dependent on these the inhibition of pRB phosphatase activity in from cells The that pRB phosphatase activity to was with that dephosphorylated pRB in cells J.W. Biol. 1995; PubMed Scopus Google Scholar). results the that and dephosphorylation of pRB in Balb/c 3T3 by PP2A but had or no effect on the direct dephosphorylation of pRB by the involved in dephosphorylation of pRB selective inhibition of the of on pRB kinase is selective for inhibition of was used due to its to cells J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). Because of roles in of on the G1 and on the G1 cyclin-dependent kinases CDK4, and Protein kinase activities were in complexes as The CDK activities from cells growing in were from cells of cells growing in with caused a decrease in the activities of pRB kinases and activity was by 1 activity was to of that in cells and was the activity in The decrease in activity was and a of a of the pRB kinase activity in Balb/c 3T3 complexes had pRB kinase activity or kinase activity was also inhibited activity was to of levels The dephosphorylation of pRB that the correlated with the decrease in activity. The decrease in kinase activities were not due to in the of CDK that of CDKs were in from and cells and of the also that the levels of CDK were not by The levels of cyclin D and cyclin were by A decrease in cyclin D was 1 the of and cyclin levels were D and levels in cells for were in cells the decreases in cyclin D and were to a decrease in CDK the of cyclin D and associated with and and were from cells the in The were with D and The of cyclin D and were in that even though caused a decrease in the levels of cyclin D and E, the levels in these asynchronously growing cells were to and also caused a in the of cyclin E. A form of cyclin that was not in cells The of was to the cell E. Science. PubMed Scopus Google Scholar) and was due to phosphorylation of cyclin M. J.M. 1996; 10: PubMed Scopus Google EMBO J. 1996; PubMed Scopus Google Scholar). Balb/c 3T3 cells Mol. Cell. Biol. 1996; PubMed Scopus Google to were by of the and was of cells with caused a decrease in cyclin D The levels of cyclin D2 were by the levels of and were in and effect of on cyclin D2 that regulating the levels of cyclin D2 were from regulating and In to on cyclin levels and CDK activity with inhibition of protein phosphatases, a for on cyclin was caused dose-dependent decreases in cyclin D levels and kinase activity. The decrease in cyclin D levels was not the was the PP2A is completely inhibited decrease in cyclin D was The decrease in kinase activity was to and occurred concentrations that had no effect on cyclin D activity was and a by The for the decrease in cyclin D and activity inhibition of PP2A and not The that activity was inhibited by concentrations that had no effect on cyclin D and that was no decrease in cyclin that the decrease in cyclin D was not involved in the inhibition of activity. The results of the and show that causes decreases in cyclin D2 and cyclin E, and inhibition of and kinase activities in Balb/c 3T3 in cyclin and CDK activity were in cells with 1 The decreases in CDK activity and pRB dephosphorylation that of cyclin D-CDK4, cyclin E-CDK2, and to a cyclin are important of phosphatase decreases in pRB phosphorylation. The of cyclin the decrease in and dephosphorylation of pRB were concentrations of and that or a to an important of pathways that the levels of cyclin D2 and cyclin E, and the activities of and In to by the availability of cyclins, cyclin and kinases are regulated by CDK inhibitors C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (5016) Google Scholar). of CDK inhibitors also to phosphatase of pRB was by the of and on of and Balb/c 3T3 cells were with and cell were with and In growing and were levels caused a in and The for induction of and were to dephosphorylation of pRB was completely dephosphorylated 1 a was no in or of cells with either actinomycin to transcription, or to completely inhibited the in and In contrast, actinomycin D had an effect on pRB phosphorylation that the in and were due to but that induction was not for pRB In to and the levels of were not by Treating cells with 1 caused in and to with also had no effect on the levels of The induction of and by was in were and a was the with decreases in cyclin D and of CDK4, the concentrations of that of and selective inhibition of The that or a is involved in pathways that of and The results of show that and PP2A are involved in of pRB phosphorylation but that play distinct roles in to the that is the pRB phosphatase in while PP2A to the activities of G1cyclin-dependent The actions of the phosphatases were inhibitors that either or of and PP2A activity in from cells the of the Okadaic acid a PP2A for and is the selective of the inhibitors M. 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The of phosphatase inhibitors on Balb/c 3T3 cells a major role for in dephosphorylation of pRB in inhibition of PP2A with either or dephosphorylation of that a pRB phosphatase active these with PP2A selective concentrations of these inhibitors had no effect on pRB phosphatase activity in from The pRB phosphatase in from or cells as on its to In to PP2A selective the in of pRB in the highly phosphorylated state and inhibited pRB phosphatase activity. was not as selective for as and were for PP2A in the of to pRB phosphatase activity was with its as The that concentrations of and that completely inhibited PP2A had no effect on pRB phosphatase that PP2A a role in the direct dephosphorylation of been as a major activity that pRB during J.W. Livingston D.M. DeCaprio J.A. Mol. Cell. Biol. PubMed Scopus Google Scholar, S. J.R. S. 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