Differential Regulation of Retinoblastoma Protein Function by Specific Cdk Phosphorylation Sites

doi:10.1074/jbc.271.14.8313

Differential Regulation of Retinoblastoma Protein Function by Specific Cdk Phosphorylation Sites (*)

Erik S. Knudsen and
Jean Y. J. Wang(§)

From the Department of Biology and Center for Molecular Genetics, University of California at San Diego, La Jolla, California 92093-0347

^§ To whom correspondence should be addressed. Tel.: 619-534-6253; Fax: 619-534-2821; jywang{at}UCSD.edu.

Abstract

The retinoblastoma tumor suppressor protein, RB, contains at least three distinct protein binding domains. The A/B pocket binds proteins with the LXCXE motif, the C pocket binds the nuclear c-Abl tyrosine kinase, and the large A/B pocket binds the transcription factor E2F. Dissociation of RB from its targets is observed as RB becomes phosphorylated during G/S progression. There are 16 Cdk consensus phosphorylation sites in RB. It was previously unknown whether the many phosphorylation sites had redundant or distinct functions in the regulation of RB. Using RB mutant proteins lacking specific phosphorylation sites, we show that each of the binding domains is inhibited by different sites. Thr-821/826 phosphorylation is required to inhibit the binding to LXCXE containing proteins. Mutation of these two sites does not interfere with the hyperphosphorylation of RB. However, this phosphorylated mutant retains the ability to bind T-Ag, E7, and Elf-1, all of which contain the LXCXE motif. In contrast, Ser-807/811 phosphorylation is required to disrupt c-Abl binding. Mutation of Ser-807/811 and Thr-821/826 does not abolish the regulation of E2F binding. Taken together, these results show that the protein binding domains of RB are each regulated by distinct Cdk phosphorylation sites.

Footnotes

↵* This work was supported by National Institutes of Health Grant CA58320 (to J. Y. J. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

T-Ag

SV40 large T-antigen

GST

glutathione S-transferase

BES

2-[bis(2-hydroxyethyl)-2-amino]ethanesulfonic acid

PAGE

polyacrylamide gel electrophoresis

FL

full-length

RSV

Rous sarcoma virus

CMV

cytomegalovirus

CycA

cyclin A

WT

wild type

ME

fragment of RB encoding amino acids 835-928.
- Received November 14, 1995.
- Revision received January 22, 1996.