Publication Date

2010-12-17

Availability

Open access

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

December 2010

First Committee Member

Thomas K. Harris, PhD - Committee Chair

Second Committee Member

Terace Fletcher, PhD - Committee Member

Third Committee Member

Kerry Burnstein, PhD - Committee Member

Fourth Committee Member

Joyce M. Slingerland, M.D., PhD - Mentor

Fifth Committee Member

Gennaro D'Urso, PhD - Outside Committee Member

Abstract

Cdk2 importantly regulates G1 progression. Cdk2 activation requires cyclin binding and phosphorylation at T160 by CAK. Here we describe a novel Cdk2 site whose Akt dependent phosphorylation appears to facilitate cyclin-Cdk2 assembly Cdk2 bears a Akt consensus motif containing threonine 39 (T39) immediately preceding the PSTAIRE helix. Cellular Cdk2 co-precipitated with Akt and Akt phosphorylated Cdk2 in vitro. Treatment of quiescent cells with serum leads to activation of the Akt pathway, followed by phosphorylation of Cdk2T39. This phosphorylation preceded the formation of cyclin-Cdk2 complexes and the phosphorylation at Cdk2T160. PI3K inhibition caused cyclin E dissociation from Cdk2, loss of CAK phosphorylated Cdk2 prior to G1 arrest. Transfected Cdk2T39A showed reduced association with cellular cyclin, whereas phosphomimetic Cdk2T39E showed an increased association with cellular cyclin. Cdk2 phosphorylation by Akt increased its ability to bind cyclins E and A in vitro. We postulate that phosphorylation at Cdk2T39 in humans or the homologous site, Cdc28S46 in S. cerevisae promotes cyclin binding and thereby regulates G-1-s timing. To test this we creased S. cerevisiae, in which the endogenous CDC28 gene is replaced by either CDC28S46A or CDC28S46E. The S46E bearing strain has a shorter G1 interval compared to the WT strain. Additionally, cdcd28S46E has higher catalytic activity throughout the cell cycle. These data support the notion that T39 phosphorylation of Cdk2 facilitates cyclin binding and subsequent CAK action.

Keywords

Awesome; Cool; Cancer; Phosphorylation; Cdk2

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