The structure of CDK4/cyclin D3 has implications for models of CDK activation

Cyclin-dependent kinase 4 (CDK4)/cyclin D complexes are expressed early in the G1 phase of the cell cycle and stimulate the expression of genes required for G1 progression by phosphorylation of the product of the retinoblastoma gene, pRb. To elaborate the molecular pathway of CDK4 activation and substrate selection we have determined the structure of nonphosphorylated CDK4/cyclin D3. This structure of an authentic CDK/cyclin complex shows that cyclin binding may not be sufficient to drive the CDK active site toward an active conformation. Phosphorylated CDK4/cyclin D3 is active as a pRb kinase and is susceptible to inhibition by p27Kip1. Unlike CDK2/cyclin A, CDK4/cyclin D3 can be inactivated by treatment with λ-phosphatase, implying that phosphorylated T172 is accessible to a generic phosphatase while bound to a cyclin. Taken together, these results suggest that the structural mechanism of CDK4/cyclin D3 activation differs markedly from that of previously studied CDK/cyclin complexes.

[1]  John Kuriyan,et al.  An Allosteric Mechanism for Activation of the Kinase Domain of Epidermal Growth Factor Receptor , 2006, Cell.

[2]  David M. Livingston,et al.  Functional interactions of the retinoblastoma protein with mammalian D-type cyclins , 1993, Cell.

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

[4]  N. Pavletich,et al.  Structural basis of inhibition of CDK-cyclin complexes by INK4 inhibitors. , 2000, Genes & development.

[5]  L. Johnson,et al.  Effects of Phosphorylation of Threonine 160 on Cyclin-dependent Kinase 2 Structure and Activity* , 1999, The Journal of Biological Chemistry.

[6]  D. Morgan,et al.  Activation of cyclin-dependent kinase 4 (cdk4) by mouse MO15-associated kinase , 1994, Molecular and cellular biology.

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

[8]  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.

[9]  Martin E M Noble,et al.  The Role of the Phospho-CDK2/Cyclin A Recruitment Site in Substrate Recognition* , 2006, Journal of Biological Chemistry.

[10]  L. Johnson,et al.  The crystal structure of human CDK7 and its protein recognition properties. , 2004, Structure.

[11]  M. Ewen,et al.  Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4. , 1993, Genes & development.

[12]  M. Noble,et al.  How Tyrosine 15 Phosphorylation Inhibits the Activity of Cyclin-dependent Kinase 2-Cyclin A* , 2007, Journal of Biological Chemistry.

[13]  T. Hunt,et al.  The Crystal Structure of Human Cyclin B , 2007, Cell cycle.

[14]  K. Henrick,et al.  Inference of macromolecular assemblies from crystalline state. , 2007, Journal of molecular biology.

[15]  S. Shurtleff,et al.  D-type cyclin-dependent kinase activity in mammalian cells , 1994, Molecular and cellular biology.

[16]  Fang Liu,et al.  Cyclin-dependent kinases regulate the antiproliferative function of Smads , 2004, Nature.

[17]  P. Sicinski,et al.  Cell Cycle Progression without Cyclin D-CDK4 and Cyclin D-CDK6 Complexes , 2005, Cell cycle.

[18]  M. Garber,et al.  The interaction between HIV-1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycT1 protein. , 1998, Genes & development.

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

[20]  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.

[21]  L. Johnson,et al.  Cyclin B and Cyclin A Confer Different Substrate Recognition Properties on CDK2 , 2007, Cell cycle.

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

[23]  L. Tsai,et al.  Structure and regulation of the CDK5-p25(nck5a) complex. , 2001, Molecular cell.

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

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

[26]  Laurence H Pearl,et al.  Hsp90 and Cdc37 -- a chaperone cancer conspiracy. , 2005, Current opinion in genetics & development.

[27]  M. Noble,et al.  Reversal of Growth Suppression by p107 via Direct Phosphorylation by Cyclin D1/Cyclin-Dependent Kinase 4 , 2002, Molecular and Cellular Biology.

[28]  Sung-Hou Kim,et al.  Structural basis for CDK6 activation by a virus-encoded cyclin , 2002, Nature Structural Biology.

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

[30]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[31]  Kornelia Polyak,et al.  Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex , 1995, Nature.

[32]  Rajiv Chopra,et al.  Crystal structure of human CDK4 in complex with a D-type cyclin , 2009, Proceedings of the National Academy of Sciences.

[33]  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.

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

[35]  Randy J Read,et al.  Electronic Reprint Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination , 2022 .

[36]  P. Jeffrey,et al.  Structural basis of cyclin-dependent kinase activation by phosphorylation , 1996, Nature Structural Biology.

[37]  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.

[38]  Sung-Hou Kim,et al.  Crystal structure of cyclin-dependent kinase 2 , 1993, Nature.

[39]  P. Fowler,et al.  Substrate Specificity of CDK2-Cyclin A , 2003, Journal of Biological Chemistry.

[40]  B. Peterlin,et al.  Controlling the elongation phase of transcription with P-TEFb. , 2006, Molecular cell.

[41]  James M. Roberts,et al.  Living with or without cyclins and cyclin-dependent kinases. , 2004, Genes & development.

[42]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[43]  L. Tsai,et al.  Structure and Regulation of the CDK5-p25nck5a Complex , 2001 .

[44]  O. Bensaude,et al.  MAQ1 and 7SK RNA Interact with CDK9/Cyclin T Complexes in a Transcription-Dependent Manner , 2003, Molecular and Cellular Biology.

[45]  C. Sherr,et al.  Regulation of cyclin D-dependent kinase 4 (cdk4) by cdk4-activating kinase , 1994, Molecular and cellular biology.

[46]  Lisa M. Stevenson,et al.  Kinetic Basis for Activation of CDK2/Cyclin A by Phosphorylation* , 2001, The Journal of Biological Chemistry.

[47]  A. Giordano,et al.  Rb family proteins as modulators of gene expression and new aspects regarding the interaction with chromatin remodeling enzymes , 2006, Oncogene.

[48]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[49]  M. Barbacid,et al.  Mammalian cyclin-dependent kinases. , 2005, Trends in biochemical sciences.

[50]  L. Johnson,et al.  The structure of P‐TEFb (CDK9/cyclin T1), its complex with flavopiridol and regulation by phosphorylation , 2008, The EMBO journal.