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Molecular and Cellular Biology, December 2000, p. 8748-8757, Vol. 20, No. 23
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.
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
*
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.
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