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 recognition by cyclin A-cdk2. This hydrophobic patch is approximately 35A away from the active site of cdk2 and contains the MRAIL sequence conserved among a number of mammalian cyclins. In the x-ray structure of cyclin A-cdk2-p27, this hydrophobic patch contacts the RNLFG sequence in p27 that is common to a number of substrates and inhibitors of mammalian cdks. We find that mutation of this hydrophobic patch on cyclin A eliminates binding to proteins containing RXL motifs without affecting binding to cdk2. This docking site is critical for cyclin A-cdk2 phosphorylation of substrates containing RXL motifs, but not for phosphorylation of histone H1. Impaired substrate binding by the cyclin is the cause of the defect in RXL substrate phosphorylation, because phosphorylation can be rescued by restoring a cyclin A-substrate interaction in a heterologous manner. In addition, the conserved hydrophobic patch is important for cyclin A function in cells, contributing to cyclin A's ability to drive cells out of the G1 phase of the cell cycle. Thus, we define a mechanism by which cyclins can recruit substrates to cdks, and our results support the notion that a high local concentration of substrate provided by a protein-protein interaction distant from the active site is critical for phosphorylation by cdks.
Massachusetts General Hospital Cancer Center, Building 149, 13th Street, Charlestown, MA 02129, USA.
B A
BA
Schulman
D L
DL
Lindstrom
E
E
Harlow
D000595:Amino Acid Sequence; D042846:CDC2-CDC28 Kinases; D002460:Cell Line; D019925:Cyclin A / Q000737:chemistry / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D019925:Cyclin A; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases