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

Abstract

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.

ACKNOWLEDGMENTS

We thank Ed Harlow for the C160 (anti-cyclin A) and pAB419 (anti-SV40 T-antigen) antibodies, Jackie Lees for the LLF4-1 (anti-E2F4) antibody, Jonathan Pines for pRcCMV cyclin A and pRcCMV cyclin E, José Ayté for invaluable assistance in the production of figures, and David Livingston and Mark Ewen and all members of the Division of Neoplastic Disease Mechanisms for many helpful comments.

P.D.A. was supported by the Novartis Pharmaceutical Corporation and the Cancer Research Foundation of America, W.R.S. was supported by an NIH Physician Scientist Award, and W.G.K. was supported by Novartis Pharmaceutical Corporation and is a Howard Hughes Medical Institute assistant investigator.