Dynamic Tyrosine Phosphorylation Modulates Cycling of the HSP90-P50CDC37-AHA1 Chaperone Machine

Abstract

SUMMARY Many critical protein kinases rely on the Hsp90 chaperone machinery for stability and function. After initially forming a ternary complex with kinase client and the co-chaperone p50Cdc37, Hsp90 proceeds through a cycle of conformational changes facilitated by ATP binding and hydrolysis. Progression through the chaperone cycle requires release of p50Cdc37 and recruitment of the ATPase activating co-chaperone AHA1, but the molecular regulation of this complex process at the cellular level is poorly understood. We demonstrate that a series of tyrosine phosphorylation events, involving both p50Cdc37 and Hsp90, are minimally sufficient to provide directionality to the chaperone cycle. p50Cdc37 phosphorylation on Y4 and Y298 disrupts client-p50Cdc37 association, while Hsp90 phosphorylation on Y197 dissociates p50Cdc37 from Hsp90. Hsp90 phosphorylation on Y313 promotes recruitment of AHA1 which stimulates Hsp90 ATPase activity, furthering the chaperoning process. Finally, at completion of the chaperone cycle, Hsp90 Y627 phosphorylation induces dissociation of the client and remaining co-chaperones.

Structural and functional basis of protein phosphatase 5 substrate specificity

“…Serine/threonine phosphorylation of Hsp90 itself is, in general, found to result in reduced affinity with clients (31)(32)(33). Client kinase release is also triggered by tyrosine phosphorylation of Cdc37 on Y298 by the Src family kinase YES, and tyrosine phosphorylation of Hsp90 on Y627 dissociates Cdk4 (21). The latter modification also dissociates Aha1 and PP5 from Hsp90 (21) and as such, may be a mechanism for terminating one cycle of chaperone activity.…”

“…Client kinase release is also triggered by tyrosine phosphorylation of Cdc37 on Y298 by the Src family kinase YES, and tyrosine phosphorylation of Hsp90 on Y627 dissociates Cdk4 (21). The latter modification also dissociates Aha1 and PP5 from Hsp90 (21) and as such, may be a mechanism for terminating one cycle of chaperone activity. Together, these events emphasize the intricacy of the client activation process and the many layers of control provided by serine/threonine and tyrosine phosphorylation and its reversal.…”

The Transcriptional Cascade in the Heat Stress Response of Arabidopsis Is Strictly Regulated at the Level of Transcription Factor Expression

“…Because the activity of Hs-HSF1 is regulated by phosphorylation (Xu et al, 2012b), we focused on Tyr-271, which is the only amino acid in the conserved motif of the TDR with the potential to be phosphorylated. We replaced Tyr-271 with phenylalanine (dY271F) or aspartic acid (dY271D) to mimic the nonphosphorylated or phosphorylated forms of the protein, respectively (Xu et al, 2012a; Figure 3A). The transactivation and DNA binding activities of dY271D were as high as those of dD1, thereby indicating that the phosphomimetic mutant of Tyr-271 had the same effect as deletion of region 1 in terms of disruption of the repressive mechanism of HsfA1d activity.…”

Aha1 Can Act as an Autonomous Chaperone to Prevent Aggregation of Stressed Proteins

“…For efficient activity the N-terminal domain of Aha1 (Aha1N) and the C-terminal domain of Aha1 associate with the Hsp90 dimer in an asymmetric fashion, contacting Hsp90M and the catalytic Hsp90N domain (8,16). Furthermore, the association between Hsp90 and the cochaperones Aha1 and Cdc37/p50 that regulate its ATP hydrolysis rate is controlled by phosphorylation of the molecular chaperone at distinct tyrosine residues during the chaperone cycle (17).…”