Dual mechanisms for the inhibition of E2F binding to RB by cyclin-dependent kinase-mediated RB phosphorylation

The growth suppression function of RB is dependent on its protein binding activity. RB contains at least three distinct protein binding functions: (i) the A/B pocket, which binds proteins with the LXCXE motif; (ii) the C pocket, which binds the c-Abl tyrosine kinase; and (iii) the large A/B pocket, which binds the E2F family of transcription factors. Phosphorylation of RB, which is catalyzed by cyclin-dependent protein kinases, inhibits all three protein binding activities. We have previously shown that LXCXE binding is inactivated by the phosphorylation of two threonines (Thr821 and Thr826), while the C pocket is inhibited by the phosphorylation of two serines (Ser807 and Ser811). In this report, we show that the E2F binding activity of RB is inhibited by two sets of phosphorylation sites acting through distinct mechanisms. Phosphorylation at several of the seven C-terminal sites can inhibit E2F binding. Additionally, phosphorylation of two serine sites in the insert domain can inhibit E2F binding, but this inhibition requires the presence of the RB N-terminal region. RB mutant proteins lacking all seven C-terminal sites and two insert domain serines can block Rat-1 cells in G1. These RB mutants can bind LXCXE proteins, c-Abl, and E2F even after they become phosphorylated at the remaining nonmutated sites. Thus, multiple phosphorylation sites regulate the protein binding activities of RB through different mechanisms, and a constitutive growth suppressor can be generated through the combined mutation of the relevant phosphorylation sites in RB.

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[2]  J. Wang.  Retinoblastoma protein in growth suppression and death protection. , 1997, Current opinion in genetics & development.

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[10]  F. C. Lucibello,et al.  Cell Cycle Regulation of E2F Site Occupation in Vivo , 1996, Science.

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[13]  P. J. Welch,et al.  Abrogation of retinoblastoma protein function by c-Abl through tyrosine kinase-dependent and -independent mechanisms , 1995, Molecular and cellular biology.

[14]  F. C. Lucibello,et al.  Cell cycle regulation of the cyclin A, cdc25C and cdc2 genes is based on a common mechanism of transcriptional repression. , 1995, The EMBO journal.

[15]  J. Nevins,et al.  Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes , 1995, Molecular and cellular biology.

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[26]  P. J. Welch,et al.  Disruption of retinoblastoma protein function by coexpression of its C pocket fragment. , 1995, Genes & development.

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[28]  K. Helin,et al.  Independent regions of adenovirus E1A are required for binding to and dissociation of E2F-protein complexes , 1993, Molecular and cellular biology.

[29]  P. J. Welch,et al.  A C-terminal protein-binding domain in the retinoblastoma protein regulates nuclear c-Abl tyrosine kinase in the cell cycle , 1993, Cell.

[30]  J. Nevins,et al.  Identification of distinct roles for separate E1A domains in disruption of E2F complexes , 1993, Molecular and cellular biology.

[31]  J. Nevins,et al.  Expression of transcription factor E2F1 induces quiescent cells to enter S phase , 1993, Nature.

[32]  W. Kaelin,et al.  E2F-1-mediated transactivation is inhibited by complex formation with the retinoblastoma susceptibility gene product. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[33]  M. Ewen,et al.  Inhibition of cell proliferation by p107, a relative of the retinoblastoma protein. , 1993, Genes & development.

[34]  E. Lam,et al.  An E2F‐binding site mediates cell‐cycle regulated repression of mouse B‐myb transcription. , 1993, The EMBO journal.

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[36]  S. Hiebert.  Regions of the retinoblastoma gene product required for its interaction with the E2F transcription factor are necessary for E2 promoter repression and pRb-mediated growth suppression , 1993, Molecular and cellular biology.

[37]  V. Garsky,et al.  Protein domains governing interactions between E2F, the retinoblastoma gene product, and human papillomavirus type 16 E7 protein , 1993, Molecular and cellular biology.

[38]  D. Livingston,et al.  Specific enzymatic dephosphorylation of the retinoblastoma protein , 1993, Molecular and cellular biology.

[39]  G. Nolan,et al.  Production of high-titer helper-free retroviruses by transient transfection. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[40]  D. Templeton,et al.  Biological function of the retinoblastoma protein requires distinct domains for hyperphosphorylation and transcription factor binding , 1992, Molecular and cellular biology.

[41]  Wen-Hwa Lee,et al.  Abrogation by c-myc of Gl phase arrest induced by RB protein but not by p53 , 1992, Nature.

[42]  R. Weinberg,et al.  Regulation of retinoblastoma protein functions by ectopic expression of human cyclins , 1992, Cell.

[43]  B. Gallie,et al.  Transcriptional repression of the E2-containing promoters EIIaE, c-myc, and RB1 by the product of the RB1 gene. , 1992, Molecular and cellular biology.

[44]  S. Weintraub,et al.  Retinoblastoma protein switches the E2F site from positive to negative element , 1992, Nature.

[45]  W. Kaelin,et al.  Identification of a growth suppression domain within the retinoblastoma gene product. , 1992, Genes & development.

[46]  J. Nevins,et al.  Adenovirus E1A, simian virus 40 tumor antigen, and human papillomavirus E7 protein share the capacity to disrupt the interaction between transcription factor E2F and the retinoblastoma gene product. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[47]  B. Gallie,et al.  Regions controlling hyperphosphorylation and conformation of the retinoblastoma gene product are independent of domains required for transcriptional repression. , 1992, Oncogene.

[48]  J. Wang,et al.  Cell cycle regulation of retinoblastoma protein phosphorylation. , 1992, Ciba Foundation symposium.

[49]  C. Anderson,et al.  The retinoblastoma protein is phosphorylated on multiple sites by human cdc2. , 1991, The EMBO journal.

[50]  Y. Qian,et al.  The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle , 1991, Cell.

[51]  R. Weinberg,et al.  Nonfunctional mutants of the retinoblastoma protein are characterized by defects in phosphorylation, viral oncoprotein association, and nuclear tethering. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[52]  W. Lee,et al.  Retinoblastoma cancer suppressor gene product is a substrate of the cell cycle regulator cdc2 kinase. , 1991, The EMBO journal.

[53]  B. Gallie,et al.  Hyperphosphorylation of the retinoblastoma gene product is determined by domains outside the simian virus 40 large-T-antigen-binding regions , 1990, Molecular and cellular biology.

[54]  M. Ewen,et al.  Definition of the minimal simian virus 40 large T antigen- and adenovirus E1A-binding domain in the retinoblastoma gene product , 1990, Molecular and cellular biology.

[55]  W. Lee,et al.  Two distinct and frequently mutated regions of retinoblastoma protein are required for binding to SV40 T antigen. , 1990, The EMBO journal.

[56]  N. Dyson,et al.  The regions of the retinoblastoma protein needed for binding to adenovirus E1A or SV40 large T antigen are common sites for mutations. , 1990, The EMBO journal.

[57]  E. Harlow,et al.  The retinoblastoma protein is phosphorylated during specific phases of the cell cycle , 1989, Cell.

[58]  Sibylle Mittnacht,et al.  Differential Phosphorylation of the Retinoblastoma Protein by G1/S Cyclin-dependent Kinases* , 1997, The Journal of Biological Chemistry.

[59]  J. Wang.  Retinoblastoma protein in growth suppression and death protection. , 1997, Current opinion in genetics & development.

[60]  J. Harper,et al.  Cyclin D1/Cdk4 regulates retinoblastoma protein-mediated cell cycle arrest by site-specific phosphorylation. , 1997, Molecular biology of the cell.

[61]  C. Sherr.  Cancer Cell Cycles , 1996, Science.

[62]  M. Kitagawa,et al.  The consensus motif for phosphorylation by cyclin D1‐Cdk4 is different from that for phosphorylation by cyclin A/E‐Cdk2. , 1996, The EMBO journal.

[63]  P. Starostik,et al.  The Rb family contains a conserved cyclin-dependent-kinase-regulated transcriptional repressor motif , 1996, Molecular and cellular biology.

[64]  R. Weinberg,et al.  TGF beta-induced growth inhibition in primary fibroblasts requires the retinoblastoma protein. , 1996, Molecular biology of the cell.

[65]  R. Weinberg,et al.  Altered cell cycle kinetics, gene expression, and G1 restriction point regulation in Rb-deficient fibroblasts , 1996, Molecular and cellular biology.

[66]  Jean Y. J. Wang,et al.  Differential Regulation of Retinoblastoma Protein Function by Specific Cdk Phosphorylation Sites (*) , 1996, The Journal of Biological Chemistry.

[67]  F. C. Lucibello,et al.  Cell Cycle Regulation of E2F Site Occupation in Vivo , 1996, Science.

[68]  P. Farnham,et al.  Introduction to the E2F family: protein structure and gene regulation. , 1996, Current topics in microbiology and immunology.

[69]  W. Sellers,et al.  A potent transrepression domain in the retinoblastoma protein induces a cell cycle arrest when bound to E2F sites. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[70]  P. J. Welch,et al.  Abrogation of retinoblastoma protein function by c-Abl through tyrosine kinase-dependent and -independent mechanisms , 1995, Molecular and cellular biology.

[71]  F. C. Lucibello,et al.  Cell cycle regulation of the cyclin A, cdc25C and cdc2 genes is based on a common mechanism of transcriptional repression. , 1995, The EMBO journal.

[72]  J. Nevins,et al.  Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes , 1995, Molecular and cellular biology.

[73]  R. Weinberg,et al.  Growth suppression by p16ink4 requires functional retinoblastoma protein. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[74]  W. Sellers,et al.  Interaction between the retinoblastoma protein and the oncoprotein MDM2 , 1995, Nature.

[75]  B. Dynlacht,et al.  Tumour-derived p16 alleles encoding proteins defective in cell-cycle inhibition , 1995, Nature.

[76]  J. Bartek,et al.  Retinoblastoma-protein-dependent cell-cycle inhibition by the tumour suppressor p16 , 1995, Nature.

[77]  C. Ingles,et al.  Direct transcriptional repression by pRB and its reversal by specific cyclins , 1995, Molecular and cellular biology.

[78]  R. Weinberg,et al.  The retinoblastoma protein and cell cycle control , 1995, Cell.

[79]  D. Livingston,et al.  Functional interaction between E2F-4 and p130: evidence for distinct mechanisms underlying growth suppression by different retinoblastoma protein family members. , 1995, Genes & development.

[80]  M. Ewen,et al.  The transcription factor E2F-1 is a downstream target of RB action , 1995, Molecular and cellular biology.

[81]  J. Seltzer,et al.  Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product , 1995, Science.

[82]  P. J. Welch,et al.  Disruption of retinoblastoma protein function by coexpression of its C pocket fragment. , 1995, Genes & development.

[83]  W. Lee,et al.  Identification of discrete structural domains in the retinoblastoma protein. Amino-terminal domain is required for its oligomerization. , 1994, The Journal of biological chemistry.

[84]  P. J. Welch,et al.  The retinoblastoma tumor suppressor protein. , 1994, Advances in cancer research.

[85]  K. Helin,et al.  Independent regions of adenovirus E1A are required for binding to and dissociation of E2F-protein complexes , 1993, Molecular and cellular biology.

[86]  P. J. Welch,et al.  A C-terminal protein-binding domain in the retinoblastoma protein regulates nuclear c-Abl tyrosine kinase in the cell cycle , 1993, Cell.

[87]  J. Nevins,et al.  Identification of distinct roles for separate E1A domains in disruption of E2F complexes , 1993, Molecular and cellular biology.

[88]  J. Nevins,et al.  Expression of transcription factor E2F1 induces quiescent cells to enter S phase , 1993, Nature.

[89]  W. Kaelin,et al.  E2F-1-mediated transactivation is inhibited by complex formation with the retinoblastoma susceptibility gene product. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[90]  M. Ewen,et al.  Inhibition of cell proliferation by p107, a relative of the retinoblastoma protein. , 1993, Genes & development.

[91]  E. Lam,et al.  An E2F‐binding site mediates cell‐cycle regulated repression of mouse B‐myb transcription. , 1993, The EMBO journal.

[92]  B. Gallie,et al.  Speculations on the roles of RB1 in tissue‐specific differentiation, tumor initiation, and tumor progression , 1993, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[93]  S. Hiebert.  Regions of the retinoblastoma gene product required for its interaction with the E2F transcription factor are necessary for E2 promoter repression and pRb-mediated growth suppression , 1993, Molecular and cellular biology.

[94]  V. Garsky,et al.  Protein domains governing interactions between E2F, the retinoblastoma gene product, and human papillomavirus type 16 E7 protein , 1993, Molecular and cellular biology.

[95]  D. Livingston,et al.  Specific enzymatic dephosphorylation of the retinoblastoma protein , 1993, Molecular and cellular biology.

[96]  G. Nolan,et al.  Production of high-titer helper-free retroviruses by transient transfection. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[97]  D. Templeton,et al.  Biological function of the retinoblastoma protein requires distinct domains for hyperphosphorylation and transcription factor binding , 1992, Molecular and cellular biology.

[98]  R. Weinberg,et al.  Regulation of retinoblastoma protein functions by ectopic expression of human cyclins , 1992, Cell.

[99]  B. Gallie,et al.  Transcriptional repression of the E2-containing promoters EIIaE, c-myc, and RB1 by the product of the RB1 gene. , 1992, Molecular and cellular biology.

[100]  S. Weintraub,et al.  Retinoblastoma protein switches the E2F site from positive to negative element , 1992, Nature.

[101]  W. Kaelin,et al.  Identification of a growth suppression domain within the retinoblastoma gene product. , 1992, Genes & development.

[102]  J. Nevins,et al.  Adenovirus E1A, simian virus 40 tumor antigen, and human papillomavirus E7 protein share the capacity to disrupt the interaction between transcription factor E2F and the retinoblastoma gene product. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[103]  B. Gallie,et al.  Regions controlling hyperphosphorylation and conformation of the retinoblastoma gene product are independent of domains required for transcriptional repression. , 1992, Oncogene.

[104]  J. Wang,et al.  Cell cycle regulation of retinoblastoma protein phosphorylation. , 1992, Ciba Foundation symposium.

[105]  Wen-Hwa Lee,et al.  Abrogation by c-myc of Gl phase arrest induced by RB protein but not by p53 , 1992, Nature.

[106]  C. Anderson,et al.  The retinoblastoma protein is phosphorylated on multiple sites by human cdc2. , 1991, The EMBO journal.

[107]  Y. Qian,et al.  The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle , 1991, Cell.

[108]  W. Lee,et al.  Retinoblastoma cancer suppressor gene product is a substrate of the cell cycle regulator cdc2 kinase. , 1991, The EMBO journal.

[109]  B. Gallie,et al.  Hyperphosphorylation of the retinoblastoma gene product is determined by domains outside the simian virus 40 large-T-antigen-binding regions , 1990, Molecular and cellular biology.

[110]  M. Ewen,et al.  Definition of the minimal simian virus 40 large T antigen- and adenovirus E1A-binding domain in the retinoblastoma gene product , 1990, Molecular and cellular biology.

[111]  W. Lee,et al.  Two distinct and frequently mutated regions of retinoblastoma protein are required for binding to SV40 T antigen. , 1990, The EMBO journal.

[112]  N. Dyson,et al.  The regions of the retinoblastoma protein needed for binding to adenovirus E1A or SV40 large T antigen are common sites for mutations. , 1990, The EMBO journal.

[113]  E. Harlow,et al.  The retinoblastoma protein is phosphorylated during specific phases of the cell cycle , 1989, Cell.