The yeast Cdc37 molecular chaperone: its association with Cak1 and its susceptibility to loss with oxidant and molybdate treatment

In eukaryotic cells the maturation and stabilization of newly-synthesised protein kinase molecules frequently requires the action of Cdc37, a molecular chaperone that will in turn often target these maturing protein kinase molecules onto another chaperone, Hsp90. Cdc37-nascent kinase associations are generally extremely transient in the environment of the living cell. Therefore they are seldom detectable by the yeast two hybrid system. This study investigated a Cdc37-kinase interaction that was detectable by two hybrid screening, the interaction of Cdc37 with the most atypical protein kinase of yeast, cyclin-dependent kinase (Cdk)-activating kinase (Cak1). It was shown that Cdc37 and Cak1 form a complex that is unaffected by Cdc37 phosphorylation status and sufficiently stable as to be isolatable by gel filtration. Formation of this Cdc37/Cak1 complex was associated with a change to the phosphorylation status of Cak1. Mass spectrometry identified two new sites of Cak1 phosphorylation, Thr27, and Ser172. Yeast strains were constructed in which the sole, essential Cak1 has either a nonphosphorylatable or a phosphomimetic amino acid at these sites. These were then analysed for altered cell cycle progression and stress-sensitivity. Molybdate treatment of yeast was shown to cause the loss of Cdc37 and the fragmentation of Hsp90 at a discrete site. Overall, the work in this thesis indicates that Cdc37 and Cak1 form a stable complex. Structural analysis of this complex may provide unique insight into how Cdc37 interacts with the N-terminal domain of protein kinases, also the unique structure of fungal Cak1 – a kinase which differs markedly from the Cak1 of human cells and is therefore potentially a promising antifungal drug target. The thesis also provides the first evidence for: (i) yeast Cak1 being regulated by phosphorylation; (ii) Cdc37 being both a determinant of and target for oxidative stress resistance in yeast; and (iii) the molybdate anion inducing in vivo loss of Cdc37 and fragmentation of Hsp90. Publications arising from this study: Millson SH, van Oosten-Hawle P, Alkuriji MA, Truman A, Siderius M, Piper PW. (2014) Cdc37 engages in stable, S14A mutation-reinforced association with the most atypical member of the yeast kinome, Cdk-activating kinase (Cak1). Cell Stress Chaperones (in press). PMID: 24452458