Reciprocal Activation by Cyclin-Dependent Kinases 2 and 7 Is Directed by Substrate Specificity Determinants outside the T Loop

Abstract

Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the metazoan CDK-activating kinase (CAK), which activates CDKs, such as CDC2 and CDK2, through phosphorylation of a conserved threonine residue in the T loop. Full activation of CDK7 requires association with a positive regulatory subunit, cyclin H, and phosphorylation of a conserved threonine residue at position 170 in its own T loop. We show that threonine-170 of CDK7 is phosphorylated in vitro by its targets, CDC2 and CDK2, which also phosphorylate serine-164 in the CDK7 T loop, a site that perfectly matches their consensus phosphorylation site. In contrast, neither CDK4 nor CDK7 itself can phosphorylate the CDK7 T loop in vitro. The ability of CDC2 or CDK2 and CDK7 to phosphorylate each other but not themselves implies that each kinase can discriminate among closely related sequences and can recognize a substrate site that diverges from its usual preferred site. To understand the basis for this paradoxical substrate specificity, we constructed a chimeric CDK with the T loop of CDK7 grafted onto the body of CDK2. Surprisingly, the hybrid enzyme, CDK2-7, was efficiently activated in cyclin A-dependent fashion by CDK7 but not at all by CDK2. CDK2-7, moreover, phosphorylated wild-type CDK7 but not CDK2. Our results suggest that the primary amino acid sequence of the T loop plays only a minor role, if any, in determining the specificity of cyclin-dependent CAKs for their CDK substrates and that protein-protein interactions involving sequences outside the T loop can influence substrate specificity both positively and negatively.

ACKNOWLEDGMENTS

This work was supported by funding from the National Institute of General Medical Sciences (to R.P.F. and D.O.M.). R.P.F. was a Scholar of the Edward Mallinckrodt, Jr. Foundation.

We thank the National Cell Culture Center for growth of HeLa cells used in the early stages of this study. We also thank Karen Lee and Julia Saiz for kind gifts of Csk1 reagents and for much advice and assistance during the course of this work. We are grateful to Alicia Russo and Nikola Pavletich (Memorial Sloan-Kettering Cancer Center) for the kind gifts of CDK2-cyclin A complexes and to Gideon Bollag (Onyx Pharmaceuticals) for CDC2-cyclin B and CDK4-cyclin D. We thank Stéphane Larochelle for critical review of the manuscript and for helpful suggestions during the course of the work. We also thank Jeff Smith for assistance in preparing the manuscript.