Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

ABSTRACT

The retinoblastoma (pRB) family of proteins includes three proteins known to suppress growth of mammalian cells. Previously we had found that growth suppression by two of these proteins, p107 and p130, could result from the inhibition of associated cyclin-dependent kinases (cdks). One important unresolved issue, however, is the mechanism through which inhibition occurs. Here we present in vivo and in vitro evidence to suggest that p107 is a bona fide inhibitor of both cyclin A-cdk2 and cyclin E-cdk2 that exhibits an inhibitory constant (K_i) comparable to that of the cdk inhibitor p21/WAF1. In contrast, pRB is unable to inhibit cdks. Further reminiscent of p21, a second cyclin-binding site was mapped to the amino-terminal portions of p107 and p130. This amino-terminal domain is capable of inhibiting cyclin-cdk2 complexes, although it is not a potent substrate for these kinases. In contrast, a carboxy-terminal fragment of p107 that contains the previously identified cyclin-binding domain serves as an excellent kinase substrate although it is unable to inhibit either kinase. Clustered point mutations suggest that the amino-terminal domain is functionally important for cyclin binding and growth suppression. Moreover, peptides spanning the cyclin-binding region are capable of interfering with p107 binding to cyclin-cdk2 complexes and kinase inhibition. Our ability to distinguish between p107 and p130 as inhibitors rather than simple substrates suggests that these proteins may represent true inhibitors of cdks.

ACKNOWLEDGMENTS

We thank S. Woo for excellent technical help and I. Sanchez, J. Ross, and K. Cai for comments on the manuscript and helpful discussions. We thank N. Dyson, E. Harlow, T. Jacks, and B. Weinberg for wild-type and nullizygous MEFs. We are grateful to E. Harlow and L. Zhu for providing plasmids and cell lines and to D. Morgan, H. Lu, W. Harper, and H. Piwinica-Worms for plasmids and baculoviruses.

This work was supported in part by a Research Project Grant (RPG-98-074-01-GMC) from the American Cancer Society and the Department of Defense (U.S. Army award no. DAMD17-96-1-6092). B.D.D. is also most grateful to E. and K. Langone and to the Damon Runyon-Walter Winchell Cancer Fund for the generous donation of a Damon Runyon Scholar Award (DRS-01).