Identification of a conserved sequence motif that promotes Cdc37 and cyclin D1 binding to Cdk4.
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.
Zhao
Qiang
Q
Department of Oncology, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA.
Boschelli
Frank
F
Caplan
Avrom J
AJ
Arndt
Kim T
KT
D000255:Adenosine Triphosphate / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000737:chemistry*; D045744:Cell Line, Tumor; D018833:Chaperonins; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D019938:Cyclin D1 / Q000737:chemistry*; D051358:Cyclin-Dependent Kinase 4; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000737:chemistry*; D029721:Drosophila Proteins / Q000737:chemistry*; D005982:Glutathione Transferase / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry; D006801:Humans; D018832:Molecular Chaperones / Q000737:chemistry*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins; D014162:Transfection
C498362:CDC37 protein, human; D018797:Cell Cycle Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D029721:Drosophila Proteins; D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; D011518:Proto-Oncogene Proteins; D019938:Cyclin D1; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D018833:Chaperonins; D005998:Glycine