SUBSTRATE RECRUITMENT TO CYCLIN-DEPENDENT-KINASE-2 BY A MULTIPURPOSE DOCKING SITE ON CYCLIN-A

An important question in the cell cycle field is how cyclin-dependent kinases (cdks) target their substrates. We have studied the role of a conserved hydrophobic patch on the surface of cyclin A in substrate re cognition by cyclin A-cdk2. This hydrophobic patch is approximate to 3 5 Angstrom away from the active site of cdk2 and contains the MRAIL se quence conserved among a number of mammalian cyclins. In the x-ray str ucture of cyclin A-cdk2-p27, this hydrophobic patch contacts the RNLFG sequence in p27 that is common to a number of substrates and inhibito rs of mammalian cdks. We find that mutation of this hydrophobic patch on cyclin A eliminates binding to proteins containing RXL motifs witho ut affecting binding to cdk2. This docking site is critical for cyclin A-cdk2 phosphorylation of substrates containing RXL motifs, but not f or phosphorylation of histone I-Il. Impaired substrate binding by the cyclin is the cause of the defect in RXL substrate phosphorylation, be cause phosphorylation can be rescued by restoring a cyclin A-substrate interaction in a heterologous manner. In addition, the conserved hydr ophobic patch is important for cyclin A function in cells, contributin g to cyclin A's ability to drive cells out of the Gr phase of the cell cycle. Thus, we define a mechanism by which cyclins can recruit subst rates to cdks, and our results support the notion that a high local co ncentration of substrate provided by a protein-protein interaction dis tant from the active site is critical for phosphorylation by cdks.