Reversal of Growth Suppression by p107 via Direct Phosphorylation by Cyclin D1/Cyclin-Dependent Kinase 4

ABSTRACT p107 functions to control cell division and development through interaction with members of the E2F family of transcription factors. p107 is phosphorylated in a cell cycle-regulated manner, and its phosphorylation leads to its release from E2F. Although it is known that p107 physically associates with E- and A-type cyclin/cyclin-dependent kinase 2 (Cdk2) complexes through a cyclin-binding RXL motif located in the spacer domain, the mechanisms underlying p107 inactivation via phosphorylation remain poorly defined. Recent genetic evidence indicates a requirement for cyclin D1/Cdk4 complexes in p107 inactivation. In this work, we provide direct biochemical evidence for the involvement of cyclin D1/Cdk4 in the inactivation of p107's growth-suppressive function. While coexpression of cyclin D1/Cdk4 can reverse the cell cycle arrest properties of p107 in Saos-2 cells, we find that p107 in which the Lys-Arg-Arg-Leu sequence of the RXL motif is replaced by four alanine residues is largely refractory to inactivation by cyclin D/Cdk4, indicating a role for this motif in p107 inactivation without a requirement for its tight interaction with cyclin D1/Cdk4. We identified four phosphorylation sites in p107 (Thr-369, Ser-640, Ser-964, and Ser-975) that are efficiently phosphorylated by Cdk4 but not by Cdk2 in vitro and are also phosphorylated in tissue culture cells. Growth suppression by p107 containing nonphosphorylatable residues in these four sites is not reversed by coexpression of cyclin D1/Cdk4. In model p107 spacer region peptides, phosphorylation of S640 by cyclin D1/Cdk4 is strictly dependent upon an intact RXL motif, but phosphorylation of this site in the absence of an RXL motif can be partially restored by replacement of S643 by arginine. This suggests that one role for the RXL motif is to facilitate phosphorylation of nonconsensus Cdk substrates. Taken together, these data indicate that p107 is inactivated by cyclin D1/Cdk4 via direct phosphorylation and that the RXL motif of p107 plays a role in its inactivation by Cdk4 in the absence of stable binding.

[1]  D. Takeda,et al.  Mutational Analysis of the Cy Motif from p21 Reveals Sequence Degeneracy and Specificity for Different Cyclin-Dependent Kinases , 2001, Molecular and Cellular Biology.

[2]  Anindya Dutta,et al.  Multiple Mechanisms Regulate Subcellular Localization of Human CDC6* , 2001, The Journal of Biological Chemistry.

[3]  J. Harper,et al.  Cyclin-dependent kinases. , 2001, Chemical reviews.

[4]  Jiri Bartek,et al.  Phosphorylation‐dependent and ‐independent functions of p130 cooperate to evoke a sustained G1 block , 2001, The EMBO journal.

[5]  F. Christians,et al.  E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis. , 2001, Genes & development.

[6]  Anindya Dutta,et al.  A Bipartite Substrate Recognition Motif for Cyclin-dependent Kinases* , 2001, The Journal of Biological Chemistry.

[7]  A. V. van Rossum,et al.  Ablation of the retinoblastoma gene family deregulates G(1) control causing immortalization and increased cell turnover under growth-restricting conditions. , 2000, Genes & development.

[8]  T. Jacks,et al.  Targeted disruption of the three Rb-related genes leads to loss of G(1) control and immortalization. , 2000, Genes & development.

[9]  J. Harbour,et al.  The Rb/E2F pathway: expanding roles and emerging paradigms. , 2000, Genes & development.

[10]  J. Qin,et al.  Cell cycle-regulated phosphorylation of p220(NPAT) by cyclin E/Cdk2 in Cajal bodies promotes histone gene transcription. , 2000, Genes & development.

[11]  J. Nevins,et al.  E2F4 and E2F5 play an essential role in pocket protein-mediated G1 control. , 2000, Molecular cell.

[12]  N. Dyson,et al.  Requirements for cell cycle arrest by p16INK4a. , 2000, Molecular cell.

[13]  T. Jacks,et al.  The retinoblastoma gene family in differentiation and development , 1999, Oncogene.

[14]  L. Johnson,et al.  The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases , 1999, Nature Cell Biology.

[15]  J. Harbour,et al.  Cdk Phosphorylation Triggers Sequential Intramolecular Interactions that Progressively Block Rb Functions as Cells Move through G1 , 1999, Cell.

[16]  James M. Roberts,et al.  CDK inhibitors: positive and negative regulators of G1-phase progression. , 1999, Genes & development.

[17]  James M. Roberts,et al.  The p21Cip1 and p27Kip1 CDK ‘inhibitors’ are essential activators of cyclin D‐dependent kinases in murine fibroblasts , 1999, The EMBO journal.

[18]  E. Lees,et al.  Cyclin D-CDK Subunit Arrangement Is Dependent on the Availability of Competing INK4 and p21 Class Inhibitors , 1999, Molecular and Cellular Biology.

[19]  W. Sellers,et al.  Retinoblastoma Protein Contains a C-terminal Motif That Targets It for Phosphorylation by Cyclin-cdk Complexes , 1999, Molecular and Cellular Biology.

[20]  J. Harper,et al.  Interaction between cyclin-dependent kinases and human papillomavirus replication-initiation protein E1 is required for efficient viral replication. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Jiri Bartek,et al.  Phosphorylation of mammalian CDC6 by Cyclin A/CDK2 regulates its subcellular localization , 1999, The EMBO journal.

[22]  B. Schulman,et al.  Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[23]  B. Dynlacht,et al.  Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor , 1998, Molecular and Cellular Biology.

[24]  N. Dyson.  The regulation of E2F by pRB-family proteins. , 1998, Genes & development.

[25]  L. Moore,et al.  Cyclin Partners Determine Pho85 Protein Kinase Substrate Specificity In Vitro and In Vivo: Control of Glycogen Biosynthesis by Pcl8 and Pcl10 , 1998, Molecular and Cellular Biology.

[26]  A. Hinnebusch,et al.  Identification of phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis. , 1998, Analytical chemistry.

[27]  J. Bartek,et al.  The retinoblastoma protein pathway in cell cycle control and cancer. , 1997, Experimental cell research.

[28]  J. Massagué,et al.  Differential Interaction of the Cyclin-dependent Kinase (Cdk) Inhibitor p27Kip1 with Cyclin A-Cdk2 and Cyclin D2-Cdk4* , 1997, The Journal of Biological Chemistry.

[29]  B. Chu,et al.  p107 and p130 Associated Cyclin A Has Altered Substrate Specificity* , 1997, The Journal of Biological Chemistry.

[30]  J. Harper,et al.  S-Phase entry upon ectopic expression of G1 cyclin-dependent kinases in the absence of retinoblastoma protein phosphorylation , 1997, Current Biology.

[31]  B. Amati,et al.  Cyclin E and c‐Myc promote cell proliferation in the presence of p16INK4a and of hypophosphorylated retinoblastoma family proteins , 1997, The EMBO journal.

[32]  Anindya Dutta,et al.  p21CIP1 and Cdc25A: competition between an inhibitor and an activator of cyclin-dependent kinases , 1997, Molecular and cellular biology.

[33]  B. Dynlacht,et al.  Copyright © 1997, American Society for Microbiology p130 and p107 Use a Conserved Domain To Inhibit Cellular , 1997 .

[34]  G. Cooper,et al.  Ras links growth factor signaling to the cell cycle machinery via regulation of cyclin D1 and the Cdk inhibitor p27KIP1 , 1997, Molecular and cellular biology.

[35]  B. Dynlacht,et al.  Specific regulation of E2F family members by cyclin-dependent kinases , 1997, Molecular and cellular biology.

[36]  S. Reed,et al.  Cyclin E-induced S phase without activation of the pRb/E2F pathway. , 1997, Genes & development.

[37]  N. Dyson,et al.  pRB and p107/p130 are required for the regulated expression of different sets of E2F responsive genes. , 1997, Genes & development.

[38]  P. Whyte,et al.  Identification of a p130 domain mediating interactions with cyclin A/cdk 2 and cyclin E/cdk 2 complexes , 1997, Oncogene.

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

[40]  J. LaBaer,et al.  New functional activities for the p21 family of CDK inhibitors. , 1997, Genes & development.

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

[42]  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.

[43]  W. Sellers,et al.  Identification of a cyclin-cdk2 recognition motif present in substrates and p21-like cyclin-dependent kinase inhibitors , 1996, Molecular and cellular biology.

[44]  M. Solomon,et al.  A Predictive Scale for Evaluating Cyclin-dependent Kinase Substrates , 1996, The Journal of Biological Chemistry.

[45]  Anindya Dutta,et al.  Cyclin-binding motifs are essential for the function of p21CIP1 , 1996, Molecular and cellular biology.

[46]  J. Pouysségur,et al.  Cyclin D1 Expression Is Regulated Positively by the p42/p44MAPK and Negatively by the p38/HOGMAPK Pathway* , 1996, The Journal of Biological Chemistry.

[47]  Philip D. Jeffrey,et al.  Crystal structure of the p27Kip1 cyclin-dependent-kinase inibitor bound to the cyclin A–Cdk2 complex , 1996, Nature.

[48]  M. Ewen,et al.  Regulation of the retinoblastoma protein-related protein p107 by G1 cyclin-associated kinases. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

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

[50]  B. Dynlacht,et al.  p107 uses a p21CIP1-related domain to bind cyclin/cdk2 and regulate interactions with E2F. , 1995, Genes & development.

[51]  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.

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

[53]  R. Kerkhoven,et al.  Regulation of the retinoblastoma protein-related p107 by G1 cyclin complexes. , 1995, Genes & development.

[54]  E. Harlow,et al.  The pRB‐related protein p107 contains two growth suppression domains: independent interactions with E2F and cyclin/cdk complexes. , 1995, The EMBO journal.

[55]  M. Kitagawa,et al.  Phosphorylation of E2F-1 by cyclin A-cdk2. , 1995, Oncogene.

[56]  C. Sherr.  G1 phase progression: Cycling on cue , 1994, Cell.

[57]  Zhou Songyang,et al.  Use of an oriented peptide library to determine the optimal substrates of protein kinases , 1994, Current Biology.

[58]  E. Karsenti,et al.  Activation of the phosphatase activity of human cdc25A by a cdk2‐cyclin E dependent phosphorylation at the G1/S transition. , 1994, The EMBO journal.

[59]  Wilhelm Krek,et al.  Negative regulation of the growth-promoting transcription factor E2F-1 by a stably bound cyclin A-dependent protein kinase , 1994, Cell.

[60]  E. Harlow,et al.  Identification of G1 kinase activity for cdk6, a novel cyclin D partner , 1994, Molecular and cellular biology.

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

[62]  Steven K. Hanks,et al.  Identification and properties of an atypical catalytic subunit (p34PSK-J3/cdk4) for mammalian D type G1 cyclins , 1992, Cell.

[63]  S. Reed,et al.  Cyclin E/cdk2 and cyclin A/cdk2 kinases associate with p107 and E2F in a temporally distinct manner. , 1992, Genes & development.

[64]  M. Ewen,et al.  The transcription factor E2F interacts with the retinoblastoma product and a p107-cyclin A complex in a cell cycle-regulated manner , 1992, Cell.

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

[66]  T. Hunt,et al.  The A‐ and B‐type cyclin associated cdc2 kinases in Xenopus turn on and off at different times in the cell cycle. , 1990, The EMBO journal.

[67]  D O Morgan,et al.  Cyclin-dependent kinases: engines, clocks, and microprocessors. , 1997, Annual review of cell and developmental biology.

[68]  T. Hunter,et al.  Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. , 1991, Methods in enzymology.