Identification of heat shock protein 90-associated 84-kDa phosphoprotein.
In eukaryotic cells, HSP90 is associated with several protein kinases and regulates their activities. HSP90 was also reported to possess an autophosphorylase activity. In this study, we examined in vitro autophosphorylation of HSP90, which was purified from chick muscle. We show that HSP90 was not phosphorylated in vitro, but an 84-kDa protein (p84) was highly phosphorylated. P84 was neither HSP90 nor its degradative product, as it was not detected by an antibody (BF4) specific to HSP90 in denaturing immunoprecipitation and Western blot analysis. Phosphorylation of a protein similar to p84 was also detected with purified human brain and HeLa HSP90, indicating that p84 is present in many different types of cells. P84 appeared to exist as large complexes, as determined by HPLC and native gel electrophoresis. Native immunoprecipitation using anti-HSP90 (BF4)-conjugated Affi-gel revealed that this phosphoprotein is specifically associated with HSP90. The interaction of p84 and HSP90 was not affected by p84 phosphorylation. In addition, p84 phosphorylation was prevented by the presence of divalent cations such as Mg(2+) and Mn(2+). In contrast, p84 phosphorylation was significantly activated by addition of exogenous Ca(2+) between 100 and 500 microM, although it was blocked by higher concentrations (>1 mM) of Ca(2+). HSP90, but not p84, could be phosphorylated by casein kinase II. Finally, p84 phosphorylation was specifically prevented by hemin, but not by other kinase inhibitors, indicating that p84 phosphorylation may be regulated by heme-regulated protein kinase.
Kim
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Department of Molecular Biology, College of Natural Sciences, Pusan National University, Pusan, 609-735, Korea.
Kang
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Kang
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HS
Kim
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D000818:Animals; D001923:Brain Chemistry; D002118:Calcium / Q000378:metabolism; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006427:Hemin / Q000378:metabolism; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation
D018841:HSP90 Heat-Shock Proteins; D010750:Phosphoproteins; D008345:Manganese; D006427:Hemin; D008274:Magnesium; D002118:Calcium