title: Thr90 phosphorylation of Hsp90α by protein kinase A regulates its chaperone machinery.
abstract: Hsp90 (heat-shock protein 90) is one of the most important molecular chaperones in eukaryotes. Hsp90 facilitates the maturation, activation or degradation of its client proteins. It is now well accepted that both ATP binding and co-chaperone association are involved in regulating the Hsp90 chaperone machinery. However, other factors such as post-translational modifications are becoming increasingly recognized as being involved in this process. Recent studies have reported that phosphorylation of Hsp90 plays an unanticipated role in this process. In the present study, we systematically investigated the impact of phosphorylation of a single residue (Thr90) of Hsp90α (pThr90-Hsp90α) on its chaperone machinery. We demonstrate that protein kinase A specifically phosphorylates Hsp90α at Thr90, and that the pThr9090-Hsp90α level is significantly elevated in proliferating cells. Thr90 phosphorylation affects the binding affinity of Hsp90α to ATP. Subsequent examination of the interactions of Hsp90α with co-chaperones reveals that Thr90 phosphorylation specifically regulates the association of a subset of co-chaperones with Hsp90α. The Hsp90α T90E phosphor-mimic mutant exhibits increased association with Aha1 (activator of Hsp90 ATPase homologue 1), p23, PP5 (protein phosphatase 5) and CHIP (C-terminus of Hsp70-interacting protein), and decreased binding affinity with Hsp70, Cdc37 (cell division cycle 37) and Hop [Hsc70 (heat-shock cognate protein 70)/Hsp90-organizing protein], whereas its interaction with FKBP52 (FK506-binding protein 4) is only moderately affected. Moreover, we find that the ability of the T90E mutant to form complexes with its clients, such as Src, Akt or PKCγ (protein kinase Cγ), is dramatically impaired, suggesting that phosphorylation affects its chaperoning activity. Taken together, the results of the present study demonstrate that Thr90 phosphorylation is actively engaged in the regulation of the Hsp90α chaperone machinery and should be a generic determinant for the cycling of Hsp90α chaperone function.
authors.affiliation: National Engineering Laboratory for Anti-tumor Protein Therapeutics, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Wang
authors.forename: Xin-an
authors.initials: XA
authors.lastname: Lu
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Song
authors.forename: Wei
authors.initials: W
authors.lastname: Zhuo
authors.forename: Lin
authors.initials: L
authors.lastname: Jia
authors.forename: Yushan
authors.initials: Y
authors.lastname: Jiang
authors.forename: Yongzhang
authors.initials: Y
authors.lastname: Luo
mesh_terms: D000255:Adenosine Triphosphate; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D018832:Molecular Chaperones; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D018841:HSP90 Heat-Shock Proteins; C533585:HSP90AA2P protein, human; D018832:Molecular Chaperones; D000255:Adenosine Triphosphate; C087857:protein kinase C gamma; D019061:src-Family Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C