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Molecular and Cellular Biology, July 2001, p. 4773-4784, Vol. 21, No. 14
Howard Hughes Medical Institute, Washington
University School of Medicine, St. Louis, Missouri 63110
Received 6 April 2001/Accepted 10 April 2001
The retinoblastoma tumor suppressor protein (pRB) negatively
regulates early-G1 cell cycle progression, in part, by
sequestering E2F transcription factors and repressing E2F-responsive
genes. Although pRB is phosphorylated on up to 16 cyclin-dependent
kinase (Cdk) sites by multiple G1 cyclin-Cdk complexes, the
active form(s) of pRB in vivo remains unknown. pRB is present as an
unphosphorylated protein in G0 quiescent cells and becomes
hypophosphorylated (~2 mol of PO4 to 1 mol of pRB) in
early G1 and hyperphosphorylated (~10 mol of
PO4 to 1 mol of pRB) in late G1 phase. Here, we
report that hypophosphorylated pRB, present in early G1,
represents the biologically active form of pRB in vivo that is
assembled with E2Fs and E1A but that both unphosphorylated pRB in
G0 and hyperphosphorylated pRB in late G1 fail
to become assembled with E2Fs and E1A. Furthermore, using transducible
dominant-negative TAT fusion proteins that differentially target
cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin
E-Cdk2 complexes, namely, TAT-p16 and TAT-dominant-negative Cdk2,
respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G1 and that cyclin E-Cdk2
complexes inactivate pRB by hyperphosphorylation in late
G1. Moreover, we found that cycling human tumor
cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion
of the p16INK4a gene, contained
hypophosphorylated pRB that was bound to E2Fs in early G1
and that E2F-responsive genes, including those for dihydrofolate
reductase and cyclin E, were transcriptionally repressed. Thus, we
conclude that, physiologically, pRB is differentially regulated by
G1 cyclin-Cdk complexes.
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.14.4773-4784.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Differential Regulation of Retinoblastoma Tumor
Suppressor Protein by G1 Cyclin-Dependent Kinase
Complexes In Vivo
*
Corresponding author. Mailing address: Howard Hughes
Medical Institute, Washington University School of Medicine, St. Louis, MO 63110. Phone: (314) 362-1722. Fax: (314) 362-1802. E-mail: dowdy{at}pathology.wustl.edu.
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