The human cell division cycle is governed by elegant regulatory pathways, many of them conserved throughout eukaryotes, which ensure that major events occur in the proper order and that progression to the next phase is delayed until defects can be corrected. Previous work indicates that Cyclin dependent kinase 2 (Cdk2) is required for initiation of DNA synthesis in human cells and that Cdk1 (Cdc2) is required for initiation of mitosis. Mounting evidence suggests that Cdk2 plays another role by fostering Cdk1 activation. In preliminary studies, a facile experimental system was established to better define the roles of Cdk2. Stable U2-OS cell clones were generated with inducible expression of wild type and dominant-negative forms of the enzyme (Cdk2-wt and Cdk2-dn, respectively). Cdk2-wt had no apparent effect on the cell division cycle, whereas Cdk2-dn primarily arrested cells in G2 phase: these cells contained replicated DNA, uncondensed chromosomes, low levels of Cdk1 kinase activity, and high levels of tyrosine-phosphorylated Cdk1, an inactive form of the enzyme. These findings solidified the evidence that Cdk2 is needed for activation of Cdk1. Further investigation identified specific effects of Cdk2-dn on the Cdk1 activators Plk1, Cdc25C, and Cdc25B and the Cdk1 inhibitor Wee1. We now report that interfering with expression of cyclin A, one of the Cdk2 activating subunits, imposed S and G2/M delays in normal human fibroblasts and qualitatively reproduced the major biochemical effects observed with Cdk2-dn induction in U2-OS cells. We therefore hypothesize that cyclin A/Cdk2 complexes play a major role in mitotic entry by antagonizing tyrosine phosphorylation of Cdk1. We propose to explore this hypothesis through four interrelated specific aims: 1) To delineate the S and G2 phase Cdk complexes that foster Cdk1 activation, 2) To determine the mechanism by which Cdk2 increases Plk1 levels and its impact on mitotic entry, 3) To determine the mechanism by which Cdk2 mediates activation of Cdc25C and Cdc25B, and 4) To determine the mechanism by which Cdk2 mediates phosphorylation of Wee1. We will accomplish these aims through systematic biochemical analysis of the effects of inhibition and depletion of the relevant factors in replicating cells. The proposed work will define functions of Cdk complexes that link the two major phases of cell replication. These functions appear to be subject to physiologic regulation by checkpoint pathways that inhibit entry into mitosis in response to DNA damage. We believe that deregulation of Cdk2, a frequent event in cancer, may contribute to genetic instability and neoplastic progression through deregulation of these functions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065514-02
Application #
6878990
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Zatz, Marion M
Project Start
2004-05-01
Project End
2008-04-30
Budget Start
2005-05-01
Budget End
2006-04-30
Support Year
2
Fiscal Year
2005
Total Cost
$293,225
Indirect Cost
Name
University of Pennsylvania
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Enders, Greg H (2010) Gauchos and ochos: a Wee1-Cdk tango regulating mitotic entry. Cell Div 5:12
Li, Changqing; Andrake, Mark; Dunbrack, Roland et al. (2010) A bifunctional regulatory element in human somatic Wee1 mediates cyclin A/Cdk2 binding and Crm1-dependent nuclear export. Mol Cell Biol 30:116-30
Enders, Greg H (2008) Expanded roles for Chk1 in genome maintenance. J Biol Chem 283:17749-52
Enders, Greg H; Maude, Shannon L (2006) Traffic safety for the cell: influence of cyclin-dependent kinase activity on genomic stability. Gene 371:1-6
Mitra, Jayashree; Enders, Greg H; Azizkhan-Clifford, Jane et al. (2006) Dual regulation of the anaphase promoting complex in human cells by cyclin A-Cdk2 and cyclin A-Cdk1 complexes. Cell Cycle 5:661-6
Maude, Shannon L; Enders, Greg H (2005) Cdk inhibition in human cells compromises chk1 function and activates a DNA damage response. Cancer Res 65:780-6
Mitra, Jayashree; Enders, Greg H (2004) Cyclin A/Cdk2 complexes regulate activation of Cdk1 and Cdc25 phosphatases in human cells. Oncogene 23:3361-7