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• W2124166107 abstract "pRB, a negative-growth regulatory protein, is a demonstrated substrate for type 1 serine/threonine protein phosphatases (PP1). In a recent report from this laboratory, we demonstrated that select forms of phosphorylated as well as hypophosphorylated pRB can be found complexed with the α-isotype of PP1 (PP1α). This complex can also be observed when PP1 is rendered catalytically dead by toxin inhibition. These data suggested to us that pRB may bind to PP1 at one or more sites other than the catalytically active one on the enzyme and that such binding may play a role other than bringing the substrate into contact with the enzyme to facilitate catalysis. To address this possibility we utilized a series of pRB deletion mutants and coprecipitation studies to map the pRB domain involved in binding to PP1. Together with competition assays using in vivoexpression of SV40 T-antigen, we show here that the carboxyl-terminal region of pRB is both necessary and sufficient for physical interaction with PP1. Subsequent biochemical analyses demonstrated inhibition of PP1 catalytic activity toward the standard substrate phosphorylasea when this enzyme is bound to pRB containing this region.K m and V max calculations revealed that pRB binds to PP1 in a non-competitive manner. These data support the notion that pRB, in addition to being a substrate for PP1, also functions as a PP1 inhibitor. The significance of this finding with respect to the functional importance of this interaction is discussed. pRB, a negative-growth regulatory protein, is a demonstrated substrate for type 1 serine/threonine protein phosphatases (PP1). In a recent report from this laboratory, we demonstrated that select forms of phosphorylated as well as hypophosphorylated pRB can be found complexed with the α-isotype of PP1 (PP1α). This complex can also be observed when PP1 is rendered catalytically dead by toxin inhibition. These data suggested to us that pRB may bind to PP1 at one or more sites other than the catalytically active one on the enzyme and that such binding may play a role other than bringing the substrate into contact with the enzyme to facilitate catalysis. To address this possibility we utilized a series of pRB deletion mutants and coprecipitation studies to map the pRB domain involved in binding to PP1. Together with competition assays using in vivoexpression of SV40 T-antigen, we show here that the carboxyl-terminal region of pRB is both necessary and sufficient for physical interaction with PP1. Subsequent biochemical analyses demonstrated inhibition of PP1 catalytic activity toward the standard substrate phosphorylasea when this enzyme is bound to pRB containing this region.K m and V max calculations revealed that pRB binds to PP1 in a non-competitive manner. These data support the notion that pRB, in addition to being a substrate for PP1, also functions as a PP1 inhibitor. The significance of this finding with respect to the functional importance of this interaction is discussed. retinoblastoma protein protein phosphatase type 1 α-isotype of PP1 PP1 catalytic subunit glutathione S-transferase amino acids polyacrylamide gel electrophoresis The nuclear phosphoprotein product of the retinoblastoma susceptibility gene, pRB,1has occupied a central position for investigations surrounding the mechanism of cell cycle progression. Numerous studies show that the growth-suppressive property of this protein is dependent upon its cell cycle stage-dependent phosphorylation state, which affects the ability of pRB to complex with other cellular and viral proteins (Refs. 1Chellappan S.P. Hiebert S. Mudryj M. Horowitz J.M. Nevins J.R. Cell. 1991; 65: 1053-1061Abstract Full Text PDF PubMed Scopus (1093) Google Scholar, 2DeCaprio J.A. Ludlow J.W. Figge J. Shew J. Huang C. Lee W. Marsilio E. Paucha E. Livingston D.M. Cell. 1988; 54: 275-283Abstract Full Text PDF PubMed Scopus (1099) Google Scholar, 3Dyson N. Howley P.T. Münger K. Harlow E. Science. 1989; 243: 934-937Crossref PubMed Scopus (2376) Google Scholar, 4Helin K. Less J.A. Vidal M. Dyson N. Harlow E. Fattaey A. Cell. 1992; 70: 337-350Abstract Full Text PDF PubMed Scopus (521) Google Scholar, 5Kato J.-Y. Matsushime H. Hiebert S.W. Ewen M.E. Sherr C.J. Genes Dev. 1993; 7: 331-342Crossref PubMed Scopus (1082) Google Scholar, 6Ludlow J.W. DeCaprio J.A. Huang C.M. Lee W.H. Paucha E. Livingston D.M. Cell. 1989; 56: 57-65Abstract Full Text PDF PubMed Scopus (368) Google Scholar, 7Münger K. Werness B.A. Dyson N. Phelps W.C. Harlow E. Howley P.M. EMBO J. 1989; 8: 4099-4105Crossref PubMed Scopus (908) Google Scholar, 8Whyte P. Bukovich K.J. Horowitz J.M. Friend S.H. Raybuck M. Weinberg R.A. Harlow E. Nature. 1988; 334: 124-129Crossref PubMed Scopus (1032) Google Scholar, reviewed in Ref. 9Taya Y. Trends Biochem. Sci. 1997; 22: 14-17Abstract Full Text PDF PubMed Scopus (234) Google Scholar). Indeed, hypophosphorylated pRB found in early- to mid-G1 is capable of sequestering and thereby functionally inactivating transcription factors necessary for cell growth. Sequential serine/threonine phosphorylation of pRB by cyclin-dependent kinases has been shown to release these factors, after which cell proliferation commences. Beginning at anaphase, pRB becomes progressively dephosphorylated (10Ludlow J.W. Shon J. Pipas J.M. Livingston D.M. DeCaprio J.A. Cell. 1990; 60: 387-396Abstract Full Text PDF PubMed Scopus (291) Google Scholar, 11Ludlow J.W. Glendening C.L. Livingston D.M. DeCaprio J.A. Mol. Cell. Biol. 1993; 13: 367-372Crossref PubMed Scopus (221) Google Scholar) and is returned to its growth-suppressive, hypophosphorylated state by the next G1 phase. Our laboratory (11Ludlow J.W. Glendening C.L. Livingston D.M. DeCaprio J.A. Mol. Cell. Biol. 1993; 13: 367-372Crossref PubMed Scopus (221) Google Scholar) as well as others (12Alberts A.S. Thornburn A.M. Shenolikar S. Mumby M.C. Feramisco J.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 388-392Crossref PubMed Scopus (141) Google Scholar) reports that this mitotic-phase hypophosphorylation of pRB results, at least in part, by the activity of one or more members of the type 1 serine/threonine protein phosphatases (PP1). The role of PP1 in cell cycle regulation has been revealed by its requirement for successful exit from mitosis (13Axton J.M. Dombradi V. Cohen P.T. Glover D.M. Cell. 1990; 63: 33-46Abstract Full Text PDF PubMed Scopus (229) Google Scholar, 14Doonan J.H. Morris N.R. Cell. 1989; 57: 987-996Abstract Full Text PDF PubMed Scopus (236) Google Scholar, 15Fernandez A. Brautigan D.L. Lamb J.C. J. Cell Biol. 1992; 116: 1421-1430Crossref PubMed Scopus (170) Google Scholar, 16Gavin A.-C. Tsukitani Y. Schorderet-Slatkine S. Exp. Cell Res. 1991; 192: 75-81Crossref PubMed Scopus (77) Google Scholar, 17Yamashita K. Yasuda H. Pines J. Yasumoto K. Nishitani H. Ohtsubo M. Hunter T. Sugimura T. Nishimoto T. EMBO J. 1990; 9: 4331-4338Crossref PubMed Scopus (246) Google Scholar).Considered together, pRB is a cell cycle regulatory protein whose function is modulated by cell cycle-dependent serine/threonine phosphorylation. pRB enters mitosis hyperphosphorylated, yet hypophosphorylated pRB is maintained through mitosis and the subsequent G1 phase. The activity of PP1, a serine/threonine protein phosphatase, appears to be involved in M-phase progression, a time during which PP1 and pRB can be found complexed together. With such an apparent change during mitosis in the pRB phosphorylation state together with the critical timing of PP1 activity for M-phase progression, defining the biochemical and structural relationship between these two cellular proteins affords a unique opportunity for understanding the role of PP1 and pRB in cell cycle regulation.Toward this goal, we have recently demonstrated that select forms of phosphorylated as well as hypophosphorylated pRB can be found complexed with the α-isotype of PP1 (PP1α) (18Tamrakar S. Mittnacht S. Ludlow J.W. Oncogene. 1999; 18: 7803-7809Crossref PubMed Scopus (19) Google Scholar). This complex can also be observed when PP1 catalytic ability is toxin-inhibited. These data suggested to us that pRB may bind to PP1 at one or more sites other than the catalytically active one on the enzyme and that such binding may play a role other than bringing the substrate into contact with the enzyme to facilitate catalysis. To address this possibility, we have undertaken a series of experiments in which we mapped the region of pRB involved in binding to PP1, tested the catalytic activity of PP1 when complexed with pRB, and analyzed the kinetics of the reaction. As presented in this report, these data support the notion that pRB, in addition to being a substrate for PP1, binds non-competitively to this enzyme. In so doing, the catalytic activity of PP1 is inhibited.DISCUSSIONThe motive behind this study comes from the observation that hypophosphorylated pRB, a presumably poor substrate for dephosphorylation by PP1, can be readily found in a complex with this enzyme. We decided to first investigate the functional significance of this interaction by utilizing a series of pRB deletion mutants and coprecipitation studies to map the pRB domain involved in PP1 binding. Previous work by others shows that the A-B pocket of pRB, which is conserved among members of the pRB family of proteins, is required for binding with LXCXE/LXSXE motif bearing proteins such as SV40 T-antigen (36Hu Q. Dyson N. Harlow E. EMBO J. 1990; 9: 1147-1155Crossref PubMed Scopus (247) Google Scholar, 37Huang H.-J.S. Wang N-P Tseng B.Y. Lee W.-H. Lee E.Y.-H.P. EMBO J. 1990; 9: 1815-1822Crossref PubMed Scopus (153) Google Scholar, 38Kaelin Jr., W.G. Ewen M.E. Livingston D.M. Mol. Cell. Biol. 1990; 10: 3761-3769Crossref PubMed Scopus (163) Google Scholar). The A-B pocket along with C-pocket in the carboxyl terminus is essential for complex formation between pRB and E2F or cyclin D (1Chellappan S.P. Hiebert S. Mudryj M. Horowitz J.M. Nevins J.R. Cell. 1991; 65: 1053-1061Abstract Full Text PDF PubMed Scopus (1093) Google Scholar, 20Kaelin Jr., W.G. Pallas D.C. DeCaprio J.A. Kaye F.J. Livingston D.M. Cell. 1991; 64: 521-532Abstract Full Text PDF PubMed Scopus (433) Google Scholar, 43Ewen M.E. Sluss H.K. Sherr C.J. Matsushime H. Kato J. Livingston D.M. Cell. 1993; 73: 487-497Abstract Full Text PDF PubMed Scopus (915) Google Scholar, 44Hiebert S.W. Mol. Cell. Biol. 1993; 13: 3384-3391Crossref PubMed Scopus (129) Google Scholar, 45Qian Y. Luckey C. Horton L. Esser M. Templeton D.J. Mol. Cell. Biol. 1992; 12: 5363-5372Crossref PubMed Scopus (126) Google Scholar, 46Qin X.Q. Chittenden T. Livingston D.M. Kaelin Jr., W.G. Genes Dev. 1992; 6: 953-964Crossref PubMed Scopus (357) Google Scholar). The C-pocket has also been shown to bind the c-Abl tyrosine kinase (47Welch P.J. Wang J.Y. Cell. 1993; 75: 779-790Abstract Full Text PDF PubMed Scopus (366) Google Scholar, 48Welch P.J. Wang J.Y. Genes Dev. 1995; 9: 31-46Crossref PubMed Scopus (97) Google Scholar). As we report here, the carboxyl-terminal region of pRB, containing amino acids 792–928, is both necessary and sufficient for physical interaction with PP1. In support of these data, a similarly sized region of pRB (aa 773–928) appears to be required for PP1 binding when assaying by a yeast two-hybrid system (33Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1296) Google Scholar). Previous studies by others have suggested that a motif comprised of the amino acids (R/K)(I/V)XF are involved in the binding of PP1-associated proteins (49Egloff M. Johnson D.F. Moorhead G. Cohen P.T. Cohen P. Barford D. EMBO J. 1997; 16: 1876-1887Crossref PubMed Scopus (528) Google Scholar). pRB sequence analysis has revealed close matches to this motif: residues 46–49 (RLEF) and residues 874–877 (KLRF). In both cases, there is a conservative substitution in the second position of I/V to L. Interestingly, the fragment of pRB (residues 1–380) that contains the amino-terminal occurrence of this motif does not complex with PP1. The significance of this observation must await further mutagenesis studies.Competition assays using in vivo expression of SV40 T-antigen indirectly supports the aforementioned fusion protein binding data while providing further insight into the intrinsic properties of PP1 needed for binding to pRB. Both T-antigen and PP1 contain LXCXE/LXSXE motifs, which are found in several, but not all, pRB-binding proteins. Proteins that use this motif generally bind to the A-B pocket region of pRB, whereas our evidence indicates that PP1 binds to the carboxyl-terminal region of pRB. The concurrent binding of PP1 and T-antigen to pRB supports the idea that these two proteins use different, non-overlapping regions on pRB to form a ternary complex. Although not directly addressed here, these data also support the notion that PP1 does not utilize this LXSXE motif in binding to pRB. Indeed, a recent report by Dick et al. (50Dick F.A. Sailhamer E. Dyson N.J. Mol. Cell. Biol. 2000; 20: 3715-3727Crossref PubMed Scopus (103) Google Scholar) supports the notion that this motif and the corresponding binding region on pRB is more important for viral oncoprotein binding than for cellular protein binding. In contrast, the aforementioned yeast two-hybrid analysis reported by others (33Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1296) Google Scholar) includes mutagenesis data, suggesting that the A-B region of pRB may also be involved in binding both PP1 and SV40 T-antigen. However, these data contain exceptions in which deletion mutagenesis of the B-region results in obliteration of T-antigen binding, whereas PP1 binding is reduced, and deletion of the carboxyl terminus obliterates PP1 binding, whereas T-antigen binding is unaffected. Perhaps reconciliation of these apparent discrepancies lies in the difference in the type and sensitivity of the experimental assays and also their use of truncated (aa 1–273), instead of full-length, T-antigen.What is the biochemical significance of pRB binding to PP1? Taking a cue from Siegert and Robbins (42Siegert J.L. Robbins P.D. Mol. Cell. Biol. 1999; 19: 846-854Crossref PubMed Scopus (24) Google Scholar), in which they reported inhibition of kinase activity when pRB is bound to the amino-terminal kinase domain of TAFII250, we tested whether PP1 catalytic activity can be altered upon pRB binding. Enzymatic analyses of pRB/PP1α complexes reveals an inhibition of catalytic activity toward the standard substrate phosphorylase a. This inhibition appears to be dependent upon an intact carboxyl-terminal region of pRB, the same region required for pRB binding to PP1. K m andV max calculations revealed that pRB binds to PP1 in a non-competitive manner. This argues against the suggestion that the observed inhibition of PP1 activity is due to pRB occupying the active site, thereby preventing dephosphorylation of other substrates. In light of these data, we would suggest that, in addition to being a substrate for PP1, pRB also functions as an inhibitor of PP1. As additional support for this suggestion, the possibility of pRB binding to PP1 and serving as a regulator was first discussed by Durfeeet al. (33Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1296) Google Scholar). This discussion followed their observation that hypophosphorylated pRB is readily detectable in a complex with PP1, whereas hyperphosphorylated pRB is not. Building upon these observations, we recently reported that the interaction between pRB and PP1 is not dependent upon PP1 being catalytically active (18Tamrakar S. Mittnacht S. Ludlow J.W. Oncogene. 1999; 18: 7803-7809Crossref PubMed Scopus (19) Google Scholar). In fact, inhibitory toxin binding to the PP1 active site does not alter the ability of hypo- or hyperphosphorylated pRB to bind this enzyme (18Tamrakar S. Mittnacht S. Ludlow J.W. Oncogene. 1999; 18: 7803-7809Crossref PubMed Scopus (19) Google Scholar). The non-competitive inhibition data presented here validates this observation.What could be the biological significance of a potential substrate also serving as a negative regulatory protein upon association with an enzyme ? As shown previously (33Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1296) Google Scholar, 21Nelson D.A. Ludlow J.W. Oncogene. 1997; 14: 2407-2415Crossref PubMed Scopus (40) Google Scholar), interaction between PP1 and pRB occurs during early to mid G1. Perhaps accumulation of the hypophosphorylated pRB facilitates complex formation with PP1 during this period of the cell cycle and signals a decrease in the need for PP1 activity directed toward pRB. Such an inhibitory effect on PP1 could be part of a negative feedback mechanism, pRB itself playing an important role in the regulation of pRB dephosphorylation. Although speculative at this point, binding of pRB to PP1 may also affect catalytic activity toward other substrates involved in cell cycle regulation. Owing to the presence of multiple phosphorylated residues on pRB, it is also likely that several modes of PP1 regulation toward pRB take place. As reported by Liu et al. (51Liu C.W. Y Wang R-H. Dohadwala M. Schonthal A.H. Villa-Moruzzi E. Berndt N. J. Biol. Chem. 1999; 274: 29470-29475Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), inactivating phosphorylation of the PP1 catalytic subunit itself occurs in a cell cycle stage-dependent manner, which no doubt also affects the phosphorylation state of pRB. With this newly reported function of pRB, we now have another avenue of pursuit in defining the function of this protein in cell cycle regulation. The nuclear phosphoprotein product of the retinoblastoma susceptibility gene, pRB,1has occupied a central position for investigations surrounding the mechanism of cell cycle progression. Numerous studies show that the growth-suppressive property of this protein is dependent upon its cell cycle stage-dependent phosphorylation state, which affects the ability of pRB to complex with other cellular and viral proteins (Refs. 1Chellappan S.P. Hiebert S. Mudryj M. Horowitz J.M. Nevins J.R. Cell. 1991; 65: 1053-1061Abstract Full Text PDF PubMed Scopus (1093) Google Scholar, 2DeCaprio J.A. Ludlow J.W. Figge J. Shew J. Huang C. Lee W. Marsilio E. Paucha E. Livingston D.M. Cell. 1988; 54: 275-283Abstract Full Text PDF PubMed Scopus (1099) Google Scholar, 3Dyson N. Howley P.T. Münger K. Harlow E. Science. 1989; 243: 934-937Crossref PubMed Scopus (2376) Google Scholar, 4Helin K. Less J.A. Vidal M. Dyson N. Harlow E. Fattaey A. Cell. 1992; 70: 337-350Abstract Full Text PDF PubMed Scopus (521) Google Scholar, 5Kato J.-Y. Matsushime H. Hiebert S.W. Ewen M.E. Sherr C.J. Genes Dev. 1993; 7: 331-342Crossref PubMed Scopus (1082) Google Scholar, 6Ludlow J.W. DeCaprio J.A. Huang C.M. Lee W.H. Paucha E. Livingston D.M. Cell. 1989; 56: 57-65Abstract Full Text PDF PubMed Scopus (368) Google Scholar, 7Münger K. Werness B.A. Dyson N. Phelps W.C. Harlow E. Howley P.M. EMBO J. 1989; 8: 4099-4105Crossref PubMed Scopus (908) Google Scholar, 8Whyte P. Bukovich K.J. Horowitz J.M. Friend S.H. Raybuck M. Weinberg R.A. Harlow E. Nature. 1988; 334: 124-129Crossref PubMed Scopus (1032) Google Scholar, reviewed in Ref. 9Taya Y. Trends Biochem. Sci. 1997; 22: 14-17Abstract Full Text PDF PubMed Scopus (234) Google Scholar). Indeed, hypophosphorylated pRB found in early- to mid-G1 is capable of sequestering and thereby functionally inactivating transcription factors necessary for cell growth. Sequential serine/threonine phosphorylation of pRB by cyclin-dependent kinases has been shown to release these factors, after which cell proliferation commences. Beginning at anaphase, pRB becomes progressively dephosphorylated (10Ludlow J.W. Shon J. Pipas J.M. Livingston D.M. DeCaprio J.A. Cell. 1990; 60: 387-396Abstract Full Text PDF PubMed Scopus (291) Google Scholar, 11Ludlow J.W. Glendening C.L. Livingston D.M. DeCaprio J.A. Mol. Cell. Biol. 1993; 13: 367-372Crossref PubMed Scopus (221) Google Scholar) and is returned to its growth-suppressive, hypophosphorylated state by the next G1 phase. Our laboratory (11Ludlow J.W. Glendening C.L. Livingston D.M. DeCaprio J.A. Mol. Cell. Biol. 1993; 13: 367-372Crossref PubMed Scopus (221) Google Scholar) as well as others (12Alberts A.S. Thornburn A.M. Shenolikar S. Mumby M.C. Feramisco J.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 388-392Crossref PubMed Scopus (141) Google Scholar) reports that this mitotic-phase hypophosphorylation of pRB results, at least in part, by the activity of one or more members of the type 1 serine/threonine protein phosphatases (PP1). The role of PP1 in cell cycle regulation has been revealed by its requirement for successful exit from mitosis (13Axton J.M. Dombradi V. Cohen P.T. Glover D.M. Cell. 1990; 63: 33-46Abstract Full Text PDF PubMed Scopus (229) Google Scholar, 14Doonan J.H. Morris N.R. Cell. 1989; 57: 987-996Abstract Full Text PDF PubMed Scopus (236) Google Scholar, 15Fernandez A. Brautigan D.L. Lamb J.C. J. Cell Biol. 1992; 116: 1421-1430Crossref PubMed Scopus (170) Google Scholar, 16Gavin A.-C. Tsukitani Y. Schorderet-Slatkine S. Exp. Cell Res. 1991; 192: 75-81Crossref PubMed Scopus (77) Google Scholar, 17Yamashita K. Yasuda H. Pines J. Yasumoto K. Nishitani H. Ohtsubo M. Hunter T. Sugimura T. Nishimoto T. EMBO J. 1990; 9: 4331-4338Crossref PubMed Scopus (246) Google Scholar). Considered together, pRB is a cell cycle regulatory protein whose function is modulated by cell cycle-dependent serine/threonine phosphorylation. pRB enters mitosis hyperphosphorylated, yet hypophosphorylated pRB is maintained through mitosis and the subsequent G1 phase. The activity of PP1, a serine/threonine protein phosphatase, appears to be involved in M-phase progression, a time during which PP1 and pRB can be found complexed together. With such an apparent change during mitosis in the pRB phosphorylation state together with the critical timing of PP1 activity for M-phase progression, defining the biochemical and structural relationship between these two cellular proteins affords a unique opportunity for understanding the role of PP1 and pRB in cell cycle regulation. Toward this goal, we have recently demonstrated that select forms of phosphorylated as well as hypophosphorylated pRB can be found complexed with the α-isotype of PP1 (PP1α) (18Tamrakar S. Mittnacht S. Ludlow J.W. Oncogene. 1999; 18: 7803-7809Crossref PubMed Scopus (19) Google Scholar). This complex can also be observed when PP1 catalytic ability is toxin-inhibited. These data suggested to us that pRB may bind to PP1 at one or more sites other than the catalytically active one on the enzyme and that such binding may play a role other than bringing the substrate into contact with the enzyme to facilitate catalysis. To address this possibility, we have undertaken a series of experiments in which we mapped the region of pRB involved in binding to PP1, tested the catalytic activity of PP1 when complexed with pRB, and analyzed the kinetics of the reaction. As presented in this report, these data support the notion that pRB, in addition to being a substrate for PP1, binds non-competitively to this enzyme. In so doing, the catalytic activity of PP1 is inhibited. DISCUSSIONThe motive behind this study comes from the observation that hypophosphorylated pRB, a presumably poor substrate for dephosphorylation by PP1, can be readily found in a complex with this enzyme. We decided to first investigate the functional significance of this interaction by utilizing a series of pRB deletion mutants and coprecipitation studies to map the pRB domain involved in PP1 binding. Previous work by others shows that the A-B pocket of pRB, which is conserved among members of the pRB family of proteins, is required for binding with LXCXE/LXSXE motif bearing proteins such as SV40 T-antigen (36Hu Q. Dyson N. Harlow E. EMBO J. 1990; 9: 1147-1155Crossref PubMed Scopus (247) Google Scholar, 37Huang H.-J.S. Wang N-P Tseng B.Y. Lee W.-H. Lee E.Y.-H.P. EMBO J. 1990; 9: 1815-1822Crossref PubMed Scopus (153) Google Scholar, 38Kaelin Jr., W.G. Ewen M.E. Livingston D.M. Mol. Cell. Biol. 1990; 10: 3761-3769Crossref PubMed Scopus (163) Google Scholar). The A-B pocket along with C-pocket in the carboxyl terminus is essential for complex formation between pRB and E2F or cyclin D (1Chellappan S.P. Hiebert S. Mudryj M. Horowitz J.M. Nevins J.R. Cell. 1991; 65: 1053-1061Abstract Full Text PDF PubMed Scopus (1093) Google Scholar, 20Kaelin Jr., W.G. Pallas D.C. DeCaprio J.A. Kaye F.J. Livingston D.M. Cell. 1991; 64: 521-532Abstract Full Text PDF PubMed Scopus (433) Google Scholar, 43Ewen M.E. Sluss H.K. Sherr C.J. Matsushime H. Kato J. Livingston D.M. Cell. 1993; 73: 487-497Abstract Full Text PDF PubMed Scopus (915) Google Scholar, 44Hiebert S.W. Mol. Cell. Biol. 1993; 13: 3384-3391Crossref PubMed Scopus (129) Google Scholar, 45Qian Y. Luckey C. Horton L. Esser M. Templeton D.J. Mol. Cell. Biol. 1992; 12: 5363-5372Crossref PubMed Scopus (126) Google Scholar, 46Qin X.Q. Chittenden T. Livingston D.M. Kaelin Jr., W.G. Genes Dev. 1992; 6: 953-964Crossref PubMed Scopus (357) Google Scholar). The C-pocket has also been shown to bind the c-Abl tyrosine kinase (47Welch P.J. Wang J.Y. Cell. 1993; 75: 779-790Abstract Full Text PDF PubMed Scopus (366) Google Scholar, 48Welch P.J. Wang J.Y. Genes Dev. 1995; 9: 31-46Crossref PubMed Scopus (97) Google Scholar). As we report here, the carboxyl-terminal region of pRB, containing amino acids 792–928, is both necessary and sufficient for physical interaction with PP1. In support of these data, a similarly sized region of pRB (aa 773–928) appears to be required for PP1 binding when assaying by a yeast two-hybrid system (33Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1296) Google Scholar). Previous studies by others have suggested that a motif comprised of the amino acids (R/K)(I/V)XF are involved in the binding of PP1-associated proteins (49Egloff M. Johnson D.F. Moorhead G. Cohen P.T. Cohen P. Barford D. EMBO J. 1997; 16: 1876-1887Crossref PubMed Scopus (528) Google Scholar). pRB sequence analysis has revealed close matches to this motif: residues 46–49 (RLEF) and residues 874–877 (KLRF). In both cases, there is a conservative substitution in the second position of I/V to L. Interestingly, the fragment of pRB (residues 1–380) that contains the amino-terminal occurrence of this motif does not complex with PP1. The significance of this observation must await further mutagenesis studies.Competition assays using in vivo expression of SV40 T-antigen indirectly supports the aforementioned fusion protein binding data while providing further insight into the intrinsic properties of PP1 needed for binding to pRB. Both T-antigen and PP1 contain LXCXE/LXSXE motifs, which are found in several, but not all, pRB-binding proteins. Proteins that use this motif generally bind to the A-B pocket region of pRB, whereas our evidence indicates that PP1 binds to the carboxyl-terminal region of pRB. The concurrent binding of PP1 and T-antigen to pRB supports the idea that these two proteins use different, non-overlapping regions on pRB to form a ternary complex. Although not directly addressed here, these data also support the notion that PP1 does not utilize this LXSXE motif in binding to pRB. Indeed, a recent report by Dick et al. (50Dick F.A. Sailhamer E. Dyson N.J. Mol. Cell. Biol. 2000; 20: 3715-3727Crossref PubMed Scopus (103) Google Scholar) supports the notion that this motif and the corresponding binding region on pRB is more important for viral oncoprotein binding than for cellular protein binding. In contrast, the aforementioned yeast two-hybrid analysis reported by others (33Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1296) Google Scholar) includes mutagenesis data, suggesting that the A-B region of pRB may also be involved in binding both PP1 and SV40 T-antigen. However, these data contain exceptions in which deletion mutagenesis of the B-region results in obliteration of T-antigen binding, whereas PP1 binding is reduced, and deletion of the carboxyl terminus obliterates PP1 binding, whereas T-antigen binding is unaffected. Perhaps reconciliation of these apparent discrepancies lies in the difference in the type and sensitivity of the experimental assays and also their use of truncated (aa 1–273), instead of full-length, T-antigen.What is the biochemical significance of pRB binding to PP1? Taking a cue from Siegert and Robbins (42Siegert J.L. Robbins P.D. Mol. Cell. Biol. 1999; 19: 846-854Crossref PubMed Scopus (24) Google Scholar), in which they reported inhibition of kinase activity when pRB is bound to the amino-terminal kinase domain of TAFII250, we tested whether PP1 catalytic activity can be altered upon pRB binding. Enzymatic analyses of pRB/PP1α complexes reveals an inhibition of catalytic activity toward the standard substrate phosphorylase a. This inhibition appears to be dependent upon an intact carboxyl-terminal region of pRB, the same region required for pRB binding to PP1. K m andV max calculations revealed that pRB binds to PP1 in a non-competitive manner. This argues against the suggestion that the observed inhibition of PP1 activity is due to pRB occupying the active site, thereby preventing dephosphorylation of other substrates. In light of these data, we would suggest that, in addition to being a substrate for PP1, pRB also functions as an inhibitor of PP1. As additional support for this suggestion, the possibility of pRB binding to PP1 and serving as a regulator was first discussed by Durfeeet al. (33Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1296) Google Scholar). This discussion followed their observation that hypophosphorylated pRB is readily detectable in a complex with PP1, whereas hyperphosphorylated pRB is not. Building upon these observations, we recently reported that the interaction between pRB and PP1 is not dependent upon PP1 being catalytically active (18Tamrakar S. Mittnacht S. Ludlow J.W. Oncogene. 1999; 18: 7803-7809Crossref PubMed Scopus (19) Google Scholar). In fact, inhibitory toxin binding to the PP1 active site does not alter the ability of hypo- or hyperphosphorylated pRB to bind this enzyme (18Tamrakar S. Mittnacht S. Ludlow J.W. Oncogene. 1999; 18: 7803-7809Crossref PubMed Scopus (19) Google Scholar). The non-competitive inhibition data presented here validates this observation.What could be the biological significance of a potential substrate also serving as a negative regulatory protein upon association with an enzyme ? As shown previously (33Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1296) Google Scholar, 21Nelson D.A. Ludlow J.W. Oncogene. 1997; 14: 2407-2415Crossref PubMed Scopus (40) Google Scholar), interaction between PP1 and pRB occurs during early to mid G1. Perhaps accumulation of the hypophosphorylated pRB facilitates complex formation with PP1 during this period of the cell cycle and signals a decrease in the need for PP1 activity directed toward pRB. Such an inhibitory effect on PP1 could be part of a negative feedback mechanism, pRB itself playing an important role in the regulation of pRB dephosphorylation. Although speculative at this point, binding of pRB to PP1 may also affect catalytic activity toward other substrates involved in cell cycle regulation. Owing to the presence of multiple phosphorylated residues on pRB, it is also likely that several modes of PP1 regulation toward pRB take place. As reported by Liu et al. (51Liu C.W. Y Wang R-H. Dohadwala M. Schonthal A.H. Villa-Moruzzi E. Berndt N. J. Biol. Chem. 1999; 274: 29470-29475Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), inactivating phosphorylation of the PP1 catalytic subunit itself occurs in a cell cycle stage-dependent manner, which no doubt also affects the phosphorylation state of pRB. With this newly reported function of pRB, we now have another avenue of pursuit in defining the function of this protein in cell cycle regulation. The motive behind this study comes from the observation that hypophosphorylated pRB, a presumably poor substrate for dephosphorylation by PP1, can be readily found in a complex with this enzyme. We decided to first investigate the functional significance of this interaction by utilizing a series of pRB deletion mutants and coprecipitation studies to map the pRB domain involved in PP1 binding. Previous work by others shows that the A-B pocket of pRB, which is conserved among members of the pRB family of proteins, is required for binding with LXCXE/LXSXE motif bearing proteins such as SV40 T-antigen (36Hu Q. Dyson N. Harlow E. EMBO J. 1990; 9: 1147-1155Crossref PubMed Scopus (247) Google Scholar, 37Huang H.-J.S. Wang N-P Tseng B.Y. Lee W.-H. Lee E.Y.-H.P. EMBO J. 1990; 9: 1815-1822Crossref PubMed Scopus (153) Google Scholar, 38Kaelin Jr., W.G. Ewen M.E. Livingston D.M. Mol. Cell. Biol. 1990; 10: 3761-3769Crossref PubMed Scopus (163) Google Scholar). The A-B pocket along with C-pocket in the carboxyl terminus is essential for complex formation between pRB and E2F or cyclin D (1Chellappan S.P. Hiebert S. Mudryj M. Horowitz J.M. Nevins J.R. Cell. 1991; 65: 1053-1061Abstract Full Text PDF PubMed Scopus (1093) Google Scholar, 20Kaelin Jr., W.G. Pallas D.C. DeCaprio J.A. Kaye F.J. Livingston D.M. Cell. 1991; 64: 521-532Abstract Full Text PDF PubMed Scopus (433) Google Scholar, 43Ewen M.E. Sluss H.K. Sherr C.J. Matsushime H. Kato J. Livingston D.M. Cell. 1993; 73: 487-497Abstract Full Text PDF PubMed Scopus (915) Google Scholar, 44Hiebert S.W. Mol. Cell. Biol. 1993; 13: 3384-3391Crossref PubMed Scopus (129) Google Scholar, 45Qian Y. Luckey C. Horton L. Esser M. Templeton D.J. Mol. Cell. Biol. 1992; 12: 5363-5372Crossref PubMed Scopus (126) Google Scholar, 46Qin X.Q. Chittenden T. Livingston D.M. Kaelin Jr., W.G. Genes Dev. 1992; 6: 953-964Crossref PubMed Scopus (357) Google Scholar). The C-pocket has also been shown to bind the c-Abl tyrosine kinase (47Welch P.J. Wang J.Y. Cell. 1993; 75: 779-790Abstract Full Text PDF PubMed Scopus (366) Google Scholar, 48Welch P.J. Wang J.Y. Genes Dev. 1995; 9: 31-46Crossref PubMed Scopus (97) Google Scholar). As we report here, the carboxyl-terminal region of pRB, containing amino acids 792–928, is both necessary and sufficient for physical interaction with PP1. In support of these data, a similarly sized region of pRB (aa 773–928) appears to be required for PP1 binding when assaying by a yeast two-hybrid system (33Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1296) Google Scholar). Previous studies by others have suggested that a motif comprised of the amino acids (R/K)(I/V)XF are involved in the binding of PP1-associated proteins (49Egloff M. Johnson D.F. Moorhead G. Cohen P.T. Cohen P. Barford D. EMBO J. 1997; 16: 1876-1887Crossref PubMed Scopus (528) Google Scholar). pRB sequence analysis has revealed close matches to this motif: residues 46–49 (RLEF) and residues 874–877 (KLRF). In both cases, there is a conservative substitution in the second position of I/V to L. Interestingly, the fragment of pRB (residues 1–380) that contains the amino-terminal occurrence of this motif does not complex with PP1. The significance of this observation must await further mutagenesis studies. Competition assays using in vivo expression of SV40 T-antigen indirectly supports the aforementioned fusion protein binding data while providing further insight into the intrinsic properties of PP1 needed for binding to pRB. Both T-antigen and PP1 contain LXCXE/LXSXE motifs, which are found in several, but not all, pRB-binding proteins. Proteins that use this motif generally bind to the A-B pocket region of pRB, whereas our evidence indicates that PP1 binds to the carboxyl-terminal region of pRB. The concurrent binding of PP1 and T-antigen to pRB supports the idea that these two proteins use different, non-overlapping regions on pRB to form a ternary complex. Although not directly addressed here, these data also support the notion that PP1 does not utilize this LXSXE motif in binding to pRB. Indeed, a recent report by Dick et al. (50Dick F.A. Sailhamer E. Dyson N.J. Mol. Cell. Biol. 2000; 20: 3715-3727Crossref PubMed Scopus (103) Google Scholar) supports the notion that this motif and the corresponding binding region on pRB is more important for viral oncoprotein binding than for cellular protein binding. In contrast, the aforementioned yeast two-hybrid analysis reported by others (33Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1296) Google Scholar) includes mutagenesis data, suggesting that the A-B region of pRB may also be involved in binding both PP1 and SV40 T-antigen. However, these data contain exceptions in which deletion mutagenesis of the B-region results in obliteration of T-antigen binding, whereas PP1 binding is reduced, and deletion of the carboxyl terminus obliterates PP1 binding, whereas T-antigen binding is unaffected. Perhaps reconciliation of these apparent discrepancies lies in the difference in the type and sensitivity of the experimental assays and also their use of truncated (aa 1–273), instead of full-length, T-antigen. What is the biochemical significance of pRB binding to PP1? Taking a cue from Siegert and Robbins (42Siegert J.L. Robbins P.D. Mol. Cell. Biol. 1999; 19: 846-854Crossref PubMed Scopus (24) Google Scholar), in which they reported inhibition of kinase activity when pRB is bound to the amino-terminal kinase domain of TAFII250, we tested whether PP1 catalytic activity can be altered upon pRB binding. Enzymatic analyses of pRB/PP1α complexes reveals an inhibition of catalytic activity toward the standard substrate phosphorylase a. This inhibition appears to be dependent upon an intact carboxyl-terminal region of pRB, the same region required for pRB binding to PP1. K m andV max calculations revealed that pRB binds to PP1 in a non-competitive manner. This argues against the suggestion that the observed inhibition of PP1 activity is due to pRB occupying the active site, thereby preventing dephosphorylation of other substrates. In light of these data, we would suggest that, in addition to being a substrate for PP1, pRB also functions as an inhibitor of PP1. As additional support for this suggestion, the possibility of pRB binding to PP1 and serving as a regulator was first discussed by Durfeeet al. (33Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1296) Google Scholar). This discussion followed their observation that hypophosphorylated pRB is readily detectable in a complex with PP1, whereas hyperphosphorylated pRB is not. Building upon these observations, we recently reported that the interaction between pRB and PP1 is not dependent upon PP1 being catalytically active (18Tamrakar S. Mittnacht S. Ludlow J.W. Oncogene. 1999; 18: 7803-7809Crossref PubMed Scopus (19) Google Scholar). In fact, inhibitory toxin binding to the PP1 active site does not alter the ability of hypo- or hyperphosphorylated pRB to bind this enzyme (18Tamrakar S. Mittnacht S. Ludlow J.W. Oncogene. 1999; 18: 7803-7809Crossref PubMed Scopus (19) Google Scholar). The non-competitive inhibition data presented here validates this observation. What could be the biological significance of a potential substrate also serving as a negative regulatory protein upon association with an enzyme ? As shown previously (33Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1296) Google Scholar, 21Nelson D.A. Ludlow J.W. Oncogene. 1997; 14: 2407-2415Crossref PubMed Scopus (40) Google Scholar), interaction between PP1 and pRB occurs during early to mid G1. Perhaps accumulation of the hypophosphorylated pRB facilitates complex formation with PP1 during this period of the cell cycle and signals a decrease in the need for PP1 activity directed toward pRB. Such an inhibitory effect on PP1 could be part of a negative feedback mechanism, pRB itself playing an important role in the regulation of pRB dephosphorylation. Although speculative at this point, binding of pRB to PP1 may also affect catalytic activity toward other substrates involved in cell cycle regulation. Owing to the presence of multiple phosphorylated residues on pRB, it is also likely that several modes of PP1 regulation toward pRB take place. As reported by Liu et al. (51Liu C.W. Y Wang R-H. Dohadwala M. Schonthal A.H. Villa-Moruzzi E. Berndt N. J. Biol. Chem. 1999; 274: 29470-29475Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), inactivating phosphorylation of the PP1 catalytic subunit itself occurs in a cell cycle stage-dependent manner, which no doubt also affects the phosphorylation state of pRB. With this newly reported function of pRB, we now have another avenue of pursuit in defining the function of this protein in cell cycle regulation." @default.
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• W2124166107 title "The Carboxyl-terminal Region of the Retinoblastoma Protein Binds Non-competitively to Protein Phosphatase Type 1α and Inhibits Catalytic Activity" @default.
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