Purification and characterization of porcine testis 90-kDa heat shock protein (HSP90) as a substrate for various protein kinases.
We purified a large quantity of HSP90 from porcine testis by hydroxylapatite (HA-HSP90) and SDS-PAGE/electroelution (eluted-HSP90) to explore the molecular mechanism of HSP90 phosphorylation affecting its metabolism. The purified HSP90 was used as an antigen to raise polyclonal antibodies in rabbits. Immunoblot analysis revealed that most purified HSP90 was HSP90alpha. Compared with the commercial anti-HSP90 antibody, the polyclonal antibody raised in this study could specifically detect the testis HSP90 and immunoprecipitate HSP90 from tissue homogenates or cell extracts. Incubation of the purified HSP90 or HSP90 immunoprecipitated from extracts of human A431 cells, Balb/c 3T3 fibroblasts, and porcine testis with [gamma-32P]ATP/Mg2+ resulted in phosphorylation of HSP90. However, the eluted-HSP90 lost its phosphorylation ability when incubated with [gamma-32P]ATP x Mg2+ alone but could be phosphorylated by various protein kinases, including PKA, CKII, kinase FA/GSK-3 alpha, and AK. The order of phosphorylation of HSP90 by these kinases is PKA = CKII > AK >> kinase FA/GSK-3 alpha.
Huang
Hsiu-Chin
HC
Division of Biotechnology, Animal Technology Institute Taiwan, ROC.
Yu
Jau-Song
JS
Tsay
Ching-Chieann
CC
Lin
Jyh-Hung
JH
Huang
San-Yuan
SY
Fang
Wen-Teh
WT
Liu
Yin-Chang
YC
Tzang
Bor-Show
BS
Lee
Wen-Chuan
WC
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008297:Male; D011494:Protein Kinases / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D013737:Testis / Q000737:chemistry*
D000596:Amino Acids; D018841:HSP90 Heat-Shock Proteins; D011494:Protein Kinases