Abstract
Hsp90β is a major chaperone involved in numerous cellular processes. Hundreds of client proteins depend on Hsp90β for proper folding and/or activity. Regulation of Hsp90β is critical to coordinate its tasks and is mediated by several post-translational modifications. Here, we focus on two phosphorylation sites located in the charged linker region of human Hsp90β, Ser226 and Ser255, which have been frequently reported but whose function remains unclear. Targeted measurements by mass spectrometry indicated that intracellular Hsp90β is highly phosphorylated on both sites (>90%). The level of phosphorylation was unaffected by various stresses (e.g., heat shock, inhibition with drugs) that impact Hsp90β activity. Mutating the two serines to alanines increased the amount of proteins interacting with Hsp90β globally and increased the sensitivity to tryptic cleavage in the C-terminal domain. Further investigation revealed that phosphorylation on Ser255 and to a lesser extent on Ser226 is decreased in the conditioned medium of cultured K562 cells, and that a non-phosphorylatable double alanine mutant was secreted more efficiently than the wild type. Overall, our results show that phosphorylation events in the charged linker regulate both the interactions of Hsp90β and its secretion, through changes in the conformation of the chaperone.
Highlights
Heat shock protein 90 (Hsp90) is an important family of highly conserved chaperones responsible for the proper folding, the stabilization, and the targeting for degradation of many proteins [1,2,3,4]
Of all the conditions tested (42 ◦ C heat shock; ganetespib treatment; 17-DMAG treatment; serum-starvation; cells grown in acidic conditions; oxidative stress by H2 O2 added to medium), none resulted in a significant alteration of the charged linker (CL)’s phosphorylation of any isoform (Table S3). These results indicate that the phosphorylation of S226 and S255 remains stable in a variety of conditions that have an impact on Hsp90 activity and expression levels
Hsp90 mostly exerts its function by binding to other proteins, we studied the impact of CL phosphorylation on the Hsp90β interactome
Summary
Heat shock protein 90 (Hsp90) is an important family of highly conserved chaperones responsible for the proper folding, the stabilization, and the targeting for degradation of many proteins [1,2,3,4]. It is involved in numerous cellular processes and Hsp regulation is critical to coordinate its activity. Regulation of Hsp chaperone function is mediated through protein-protein interactions and post-translational modifications (PTMs) [8,9,10,11]. A complex set of PTMs is known to occur on Hsp which, together, concur to modulate its interactions and activity, resulting in a “chaperone code” analog to the one known for histones [13]
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