Functional Inactivation of the Retinoblastoma Protein Requires Sequential Modification by at Least Two Distinct Cyclin-cdk Complexes

ABSTRACT

The retinoblastoma protein (pRb) acts to constrain the G₁-S transition in mammalian cells. Phosphorylation of pRb in G₁ inactivates its growth-inhibitory function, allowing for cell cycle progression. Although several cyclins and associated cyclin-dependent kinases (cdks) have been implicated in pRb phosphorylation, the precise mechanism by which pRb is phosphorylated in vivo remains unclear. By inhibiting selectively either cdk4/6 or cdk2, we show that endogenous D-type cyclins, acting with cdk4/6, are able to phosphorylate pRb only partially, a process that is likely to be completed by cyclin E-cdk2 complexes. Furthermore, cyclin E-cdk2 is unable to phosphorylate pRb in the absence of prior phosphorylation by cyclin D-cdk4/6 complexes. Complete phosphorylation of pRb, inactivation of E2F binding, and activation of E2F transcription occur only after sequential action of at least two distinct G₁cyclin kinase complexes.

ACKNOWLEDGMENTS

We thank members of the Weinberg lab, MIT Center for Cancer Research, B. Dynlacht, S. Mittnacht, G. Paradis, and P. Utz for advice and assistance, J. Gribben, B. Dynlacht, S. van den Heuvel, W. Krek, and H. Huber for reagents, and M. Meyerson and R. Beijersbergen for critical reading of the manuscript.

R.A.W. is an American Cancer Society Professor of Biology. A.S.L. is a Margaret and Herman Sokol postdoctoral fellow and was a Howard Hughes Medical Institute Physician postdoctoral fellow. This work was supported by NIH grant K11-CA69242.