Identification of a Conserved Sequence Motif That Promotes Cdc37 and Cyclin D1 Binding to Cdk4

Abstract

Cdc37 is a molecular chaperone that is important for the stability and activity of several protein kinases, including Cdk4 and Raf1. We first determined, using in vitro assays, that Cdc37 binds to the amino-terminal lobe of Cdk4. Subsequent mutagenesis revealed that Gly-15 (G15A) and Gly-18 (G18A) were critical for Cdc37-Cdk4 complex formation. Gly-15 and Gly-18 of Cdk4 are within the conserved Gly-X-Gly-X-X-Gly motif that is required for ATP binding to the kinase. Mutation of either glycine at the equivalent positions of Raf1 (G358A and G361A) also inhibited Cdc37 binding to Raf1. Replacing another conserved residue critical for ATP binding and kinase activity, Lys-35 (K35A), reduced Cdc37-Cdk4 complex formation but to a lesser extent. The interaction of Cdk4 with Cdc37 in vitro was not sensitive to changes in ATP levels. Cell-based assays indicated that Cdk4 G15A and Cdk4 G18A were present at the same level as wild type Cdk4. Equivalent amounts of p16 bound to Cdk4 G15A and Cdk4 G18A relative to wild type Cdk4, suggesting that Cdk4 G15A and Cdk4 G18A adopt significant tertiary structure. However, in contrast to wild type Cdk4, Cdk4 G15A , and Cdk4 G18A had greatly reduced binding of cyclin D1, Cdc37, and Hsp90. Importantly, overexpression of Cdc37 not only stimulated cyclin D1 binding to wild type Cdk4 but also restored its binding to Cdk4 G15A . Under the same conditions, p16 binding to wild type Cdk4 was suppressed. Our findings show that the interaction of Cdc37 with its client protein kinases requires amino acid residues within a motif that is present in many protein kinases.

Definition of Protein Kinase Sequence Motifs That Trigger High Affinity Binding of Hsp90 and Cdc37

“…However, our results differ from those obtained in the studies utilizing the Cdk4 kinase (12). Single layered ␤-sheet structures are not stable, because the hydrophobic face of the sheet in solution would be exposed to water.…”

“…The results presented here together with previously published data (12,24) localize the Cdc37 recognition motif to the ␣-C-helix and the ␣C-␤4 loop motif in the NL of the catalytic domain of protein kinases. Cdc37 did not interact with the linker-CL Lck construct, whereas Hsp90 bound the construct.…”

“…Deletion of the ␣-C-helix negated the interaction of Cdc37, but not Hsp90 with Lck constructs containing the complete CL. Previous work investigating the interaction of Cdc37 with Cdk4 constructs fused to the C terminus of glutathione S-transferase indicated that GST-Cdk4 constructs containing the NL of Cdk4 through the ␣-C-helix and residues within the loop between the ␣-C-helix and ␤4 sheet bound Hsp90 and Cdc37 (12). Deletion of residues in the loop caused a marked reduction in Hsp90/ Cdc37 binding.…”

“…Furthermore, the domain structure of Cdc37 has been determined, and its kinase binding and Hsp90 binding activities have been mapped to its N-terminal and middle domains, respectively (20 -22), and we have demonstrated that phosphorylation of Cdc37 at Ser 13 by casein kinase II is require for the ability of Cdc37 to bind protein kinase (23). However, although the specific basis for the recognition of protein kinases by Cdc37 is unknown, recent studies have mapped the recognition motif to the N-terminal lobe of the catalytic domain of protein kinases (12,24,25).…”

“…Studies using site-directed and deletion mutagenesis (5)(6)(7)(8)(9)(10)(11) have identified potential sites within steroid hormone receptors and protein kinases that may be recognized by Hsp90. However, although a recent study has indicted that point mutations generated in the Cdk4 kinase (12) affect its interaction with Hsp90, no common primary, secondary, and/or tertiary structural motifs have been defined that may be responsible for Hsp90 client recognition.…”