Protein kinase ckii and cdc37 constitute a positive feedback loop that maintains multiple protein kinases
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Cdc37 is a protein kinase-specific chaperone that either acts by itself or in concert with Hsp90. Cdc37 is phosphorylated by the evolutionarily conserved, ubiquitously distributed Ser/Thr kinase, CKII; and this dissertation describes efforts to analyze the importance of Cdc37 phosphorylation by CKII and its potential role in regulating Cdc37 function in vivo. Non-phosphorylatable (cdc37-S14AS17A), semi-phosphorylatable (cdc37-S14A), cdc37-S17A), and quasi-phosphorylated (cdc37-S14ES17E) alleles of CDC37 were constructed and characterized. cdc37-S14AS17A was the most severely affected of all alleles and displayed a semi-lethal phenotype underscoring the importance of the CKII phosphorylation sites. cdc37-S14A was more severely affected than cdc37-S17A mutants, supporting the idea that the evolutionarily conserved CKII site at serine- 14 is more important in regulating Cdc37 function than serine-17. cdc37-S14A arrested with reduced CKII activity, and overexpression plasmids expressing cdc37- S14AS17A or cdc37M2-S14A were incapable of protecting cka2-13 mutants on media containing Geldanamycin (GA), an Hsp90-specific inhibitor. Incubation of cka2-13 cells with GA results in reduced phosphorylation of Fpr3 by CKII. Additionally, CKII activity was found to peak during G1 and G2/M phases of the cell cycle, the same phases during which Cdc37 function is essential. Genetic assays were used to demonstrate that the positive feedback loop between CKII and Cdc37 also maintains the activity of multiple protein kinases (and hence their signal transduction pathways), in addition to CKII. Overexpression of the homologous gene pair, ZDS1/2 was found to augment the function of both CKII and Cdc37, thus offering a potential mechanism for the observed pleiotropism of Zds1/2 since the feedback loop between CKII and Cdc37 regulates diverse pathways.