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Molecular and Cellular Biology, December 2000, p. 8748-8757, Vol. 20, No. 23
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Analysis of Cyclin D3-cdk4 Complexes in Fibroblasts Expressing and Lacking p27kip1 and p21cip1

Tapan Kumar Bagui,1,2 Rosalind J. Jackson,1,2 Deepak Agrawal,1,2 and W. J. Pledger1,2,3,*

Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute,1 and Department of Oncology2 and Department of Biochemistry and Molecular Biology,3 University of South Florida College of Medicine, Tampa, Florida

Received 12 June 2000/Returned for modification 17 July 2000/Accepted 14 September 2000

Our studies examined the effects of p27kip1 and p21cip1 on the assembly and activity of cyclin D3-cdk4 complexes and determined the composition of the cyclin D3 pool in cells containing and lacking these cyclin-dependent kinase inhibitors. We found that catalytically active cyclin D3-cdk4 complexes were present in fibroblasts derived from p27kip1-p21cip1-null mice and that immunodepletion of extracts of wild-type cells with antibody to p27kip1 and/or p21cip1 removed cyclin D3 protein but not cyclin D3-associated activity. Similar results were observed in experiments assaying cyclin D1-cdk4 activity. Data obtained using mixed cell extracts demonstrated that p27kip1 interacted with cyclin D3-cdk4 complexes in vitro and that this interaction was paralleled by a loss of cyclin D3-cdk4 activity. In p27kip1-p21cip1-deficient cells, the cyclin D3 pool consisted primarily of cyclin D3 monomers, whereas in wild-type cells, the majority of cyclin D3 molecules were complexed to cdk4 and either p27kip1 or p21cip1 or were monomeric. We conclude that neither p27kip1 nor p21cip1 is required for the formation of cyclin D3-cdk4 complexes and that cyclin D3-cdk4 complexes containing p27kip1 or p21cip1 are inactive. We suggest that only a minor portion of the total cyclin D3 pool accounts for all of the cyclin D3-cdk4 activity in the cell regardless of whether the cell contains p27kip1 and p21cip1.

* Corresponding author. Mailing address: H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Dr., Tampa, FL 33612. Phone: (813) 979-3887. Fax: (813) 979-3893. E-mail: pledger{at}moffitt.usf.edu.

Molecular and Cellular Biology, December 2000, p. 8748-8757, Vol. 20, No. 23
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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