p27 is regulated independently of Skp2 in the absence of Cdk2.

Cyclin-dependent kinase 2 (Cdk2) is dispensable for mitotic cell cycle progression and Cdk2 knockout mice are viable due to the compensatory functions of other Cdks. In order to assess the role of Cdk2 under limiting conditions, we used Skp2 knockout mice that exhibit increased levels of Cdk inhibitor, p27(Kip1), which is able to inhibit Cdk2 and Cdk1. Knockdown of Cdk2 abrogated proliferation of Skp2(-/-) mouse embryonic fibroblasts, encouraging us to generate Cdk2(-/-)Skp2(-/-) double knockout mice. Cdk2(-/-)Skp2(-/-) double knockout mice are viable and display similar phenotypes as Cdk2(-/-) and Skp2(-/-) mice. Unexpectedly, fibroblasts generated from Cdk2(-/-)Skp2(-/-) double knockout mice proliferated at normal rates. The increased stability of p27 observed in Skp2(-/-) MEFs was not observed in Cdk2(-/-)Skp2(-/-) double knockout fibroblasts indicating that in the absence of Cdk2, p27 is regulated by Skp2-independent mechanisms. Ablation of other ubiquitin ligases for p27 such as KPC1, DDB1, and Pirh2 did not restore stability of p27 in Cdk2(-/-)Skp2(-/-) MEFs. Our findings point towards novel and alternate pathways for p27 regulation.

[1]  S. Reed,et al.  The pRb-related protein p130 is regulated by phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCF(Skp2). , 2002, Genes & development.

[2]  C. McCall,et al.  Targeted ubiquitination of CDT1 by the DDB1–CUL4A–ROC1 ligase in response to DNA damage , 2004, Nature Cell Biology.

[3]  Xiaohua Wu,et al.  The SCFSkp2 Ubiquitin Ligase Complex Interacts with the Human Replication Licensing Factor Cdt1 and Regulates Cdt1 Degradation* , 2003, Journal of Biological Chemistry.

[4]  K. Nakayama,et al.  The F-box protein Skp2 participates in c-Myc proteosomal degradation and acts as a cofactor for c-Myc-regulated transcription. , 2003, Molecular cell.

[5]  Michele Pagano,et al.  SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27 , 1999, Nature Cell Biology.

[6]  P. Kaldis,et al.  Cdc2–cyclin E complexes regulate the G1/S phase transition , 2005, Nature Cell Biology.

[7]  M. Manns,et al.  Skp2-dependent degradation of p27kip1 is essential for cell cycle progression. , 2004, Genes & development.

[8]  C Roskelley,et al.  A biomarker that identifies senescent human cells in culture and in aging skin in vivo. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[9]  James M. Roberts,et al.  Cyclin E-CDK2 is a regulator of p27Kip1. , 1997, Genes & development.

[10]  P. Raychaudhuri,et al.  The Xeroderma Pigmentosum Group E Gene Product DDB2 Is a Specific Target of Cullin 4A in Mammalian Cells , 2001, Molecular and Cellular Biology.

[11]  M. Pagano,et al.  Role of the SCFSkp2 Ubiquitin Ligase in the Degradation of p21Cip1 in S Phase* , 2003, Journal of Biological Chemistry.

[12]  J. Massagué,et al.  Cyclic AMP-induced G1 phase arrest mediated by an inhibitor (p27 Kip1 ) of cyclin-dependent kinase 4 activation , 1994, Cell.

[13]  L. Hengst,et al.  ELAV/Hu proteins inhibit p27 translation via an IRES element in the p27 5'UTR. , 2002, Genes & development.

[14]  Keiichi I Nakayama,et al.  Molecular Dissection of the Interaction between p27 and Kip1 Ubiquitylation-promoting Complex, the Ubiquitin Ligase That Regulates Proteolysis of p27 in G1 Phase* , 2005, Journal of Biological Chemistry.

[15]  James M. Roberts,et al.  p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. , 1994, Genes & development.

[16]  Martin Wasser,et al.  Cyclin-dependent kinase 1 (Cdk1) is essential for cell division and suppression of DNA re-replication but not for liver regeneration , 2012, Proceedings of the National Academy of Sciences.

[17]  M. Kitagawa,et al.  Targeted disruption of Skp2 results in accumulation of cyclin E and p27Kip1, polyploidy and centrosome overduplication , 2000, The EMBO journal.

[18]  James M. Roberts,et al.  The murine gene p27Kip1 is haplo-insufficient for tumour suppression , 1998, Nature.

[19]  Yuhuan Zheng,et al.  Far upstream element binding protein 1 activates translation of p27Kip1 mRNA through its internal ribosomal entry site. , 2011, The international journal of biochemistry & cell biology.

[20]  Andrea C. Carrano,et al.  Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation. , 1999, Genes & development.

[21]  M. Kitagawa,et al.  Pirh2 promotes ubiquitin-dependent degradation of the cyclin-dependent kinase inhibitor p27Kip1. , 2007, Cancer research.

[22]  James M. Roberts,et al.  lnterleukin-2-mediated elimination of the p27Kipl cyclin-dependent kinase inhibitor prevented by rapamycin , 1994, Nature.

[23]  P. Kaldis,et al.  Cdk2 Knockout Mice Are Viable , 2003, Current Biology.

[24]  P. Raychaudhuri,et al.  Cullin 4A Associates with the UV-damaged DNA-binding Protein DDB* , 1999, The Journal of Biological Chemistry.

[25]  K. Nakayama,et al.  Degradation of p57Kip2 mediated by SCFSkp2-dependent ubiquitylation , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[26]  R. Elkon,et al.  A Pumilio-induced RNA structure switch in p27-3′ UTR controls miR-221 and miR-222 accessibility , 2010, Nature Cell Biology.

[27]  Wilhelm Krek,et al.  p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells , 1999, Nature Cell Biology.

[28]  B. Stillman,et al.  Human origin recognition complex large subunit is degraded by ubiquitin-mediated proteolysis after initiation of DNA replication. , 2002, Molecular cell.

[29]  Pierre Dubus,et al.  Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice , 2003, Nature Genetics.

[30]  P. Kaldis,et al.  p21 Inhibits Cdk1 in the absence of Cdk2 to maintain the G1/S phase DNA damage checkpoint. , 2008, Molecular biology of the cell.

[31]  P. Raychaudhuri,et al.  Cul4A and DDB1 Associate with Skp2 To Target p27Kip1 for Proteolysis Involving the COP9 Signalosome , 2006, Molecular and Cellular Biology.

[32]  L. Vardy,et al.  AMD1 is essential for ESC self-renewal and is translationally down-regulated on differentiation to neural precursor cells. , 2012, Genes & development.

[33]  K. Nakayama,et al.  Degradation of p27 Kip1 at the G0-G1 Transition Mediated by a Skp2-independent Ubiquitination Pathway* , 2001, The Journal of Biological Chemistry.

[34]  Nobuyuki Shishido,et al.  Mice Lacking p27 Kip1 Display Increased Body Size, Multiple Organ Hyperplasia, Retinal Dysplasia, and Pituitary Tumors , 1996, Cell.

[35]  K. Manova-Todorova,et al.  Enhanced Growth of Mice Lacking the Cyclin-Dependent Kinase Inhibitor Function of p27 Kip1 , 1996, Cell.

[36]  James M. Roberts,et al.  Cloning of p27 Kip1 , a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals , 1994, Cell.

[37]  K. Nakayama,et al.  Cytoplasmic ubiquitin ligase KPC regulates proteolysis of p27Kip1 at G1 phase , 2004, Nature Cell Biology.

[38]  H. Green,et al.  QUANTITATIVE STUDIES OF THE GROWTH OF MOUSE EMBRYO CELLS IN CULTURE AND THEIR DEVELOPMENT INTO ESTABLISHED LINES , 1963, The Journal of cell biology.

[39]  Tony Hunter,et al.  p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21 , 1994, Cell.