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Molecular and Cellular Biology, February 1999, p. 1068-1080, Vol. 19, No. 2
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Retinoblastoma Protein Contains a C-terminal Motif That Targets It for Phosphorylation by Cyclin-cdk Complexes

Peter D. Adams,1 Xiaotong Li,1 William R. Sellers,1 Kayla B. Baker,1,2 Xiaohong Leng,3 J. Wade Harper,3 Yoichi Taya,4 and William G. Kaelin Jr.1,2,*

Department of Adult Oncology1 and Howard Hughes Medical Institute,2 Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115; Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 770303; and Biology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104, Japan4

Received 30 June 1998/Returned for modification 22 July 1998/Accepted 4 November 1998

Stable association of certain proteins, such as E2F1 and p21, with cyclin-cdk2 complexes is dependent upon a conserved cyclin-cdk2 binding motif that contains the core sequence ZRXL, where Z and X are usually basic. In vitro phosphorylation of the retinoblastoma tumor suppressor protein, pRB, by cyclin A-cdk2 and cyclin E-cdk2 was inhibited by a short peptide spanning the cyclin-cdk2 binding motif present in E2F1. Examination of the pRB C terminus revealed that it contained sequence elements related to ZRXL. Site-directed mutagenesis of one of these sequences, beginning at residue 870, impaired the phosphorylation of pRB in vitro. A synthetic peptide spanning this sequence also inhibited the phosphorylation of pRB in vitro. pRB C-terminal truncation mutants lacking this sequence were hypophosphorylated in vitro and in vivo despite the presence of intact cyclin-cdk phosphoacceptor sites. Phosphorylation of such mutants was restored by fusion to the ZRXL-like motif derived from pRB or to the ZRXL motifs from E2F1 or p21. Phospho-site-specific antibodies revealed that certain phosphoacceptor sites strictly required a C-terminal ZRXL motif whereas at least one site did not. Furthermore, this residual phosphorylation was sufficient to inactivate pRB in vivo, implying that there are additional mechanisms for directing cyclin-cdk complexes to pRB. Thus, the C terminus of pRB contains a cyclin-cdk interaction motif of the type found in E2F1 and p21 that enables it to be recognized and phosphorylated by cyclin-cdk complexes.

* Corresponding author. Mailing address: Department of Adult Oncology and Howard Hughes Medical Institute, Dana-Farber Cancer Institute and Harvard Medical School, 44 Binney St., Mayer Building Room 457, Boston, MA 02115. Phone: (617) 632-3975. Fax: (617) 632-4760. E-mail: william_kaelin{at}dfci.harvard.edu.

Molecular and Cellular Biology, February 1999, p. 1068-1080, Vol. 19, No. 2
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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