Hsc70 Regulates Accumulation of Cyclin D1 and Cyclin D1-Dependent Protein Kinase

Abstract

The cyclin D-dependent kinase is a critical mediator of mitogen-dependent G₁ phase progression in mammalian cells. Given the high incidence of cyclin D1 overexpression in human neoplasias, the nature and complexity of cyclin D complexes in vivo have been subjects of intense interest. Besides its catalytic partner, the nature and complexity of cyclin D complexes in vivo remain ambiguous. To address this issue, we purified native cyclin D1 complexes from proliferating mouse fibroblasts by affinity chromatography and began to identify and functionally characterize the associated proteins. In this report, we describe the identification of Hsc70 and its functional importance for cyclin D1 and cyclin D1-dependent kinase maturation. We demonstrate that Hsc70 associates with newly synthesized cyclin D1 and is a component of a mature, catalytically active cyclin D1/CDK4 holoenzyme complex. Our data suggest that Hsc70 promotes stabilization of newly synthesized cyclin D1, thereby increasing its availability for assembly with CDK4. In addition, our data demonstrate that Hsc70 remains bound to cyclin D1 following its assembly with CDK4 and Cip/Kip proteins, where it ensures the formation of a catalytically active complex.

ACKNOWLEDGMENTS

We thank Richard Morimoto for providing the Hsc70 cDNA, David Smith for the Hsc70K71E cDNA, Greg Enders for providing the CDK2 cDNA, and Bruce Clurman for Myc-tagged cyclin E.

We acknowledge support from the American Cancer Society (grant RPG-00-303-01), National Institutes of Health (grant CA93237), the V Foundation (J.A.D.), and the National Science and Engineering Research Council of Canada (A.E.).