Heat shock proteins 70 and 90 increase calcineurin activity in vitro through calmodulin-dependent and independent mechanisms.
We have shown that heat shock proteins (HSPs) associated with steroid receptor complexes are involved in the activation of calcineurin by aldosterone and dexamethasone. To determine whether HSPs directly interact with calcineurin, we measured the effect of HSPs 90, 70 and 56 on calcineurin activity in a cell-free, in vitro system using a calcineurin-specific substrate. HSP-90 (75 or 100 nM) significantly increased calcineurin V(max) in the presence of calmodulin, while maximal stimulation by HSP-70 occurred at 50 nM. Bovine serum albumin (BSA) and actin did not change basal calcineurin activity indicating that HSP-90 and HSP-70 specifically activate calcineurin. Neither HSP-70, HSP-56, nor ATP augmented HSP-90-induced activation of calcineurin. In the absence of calmodulin, HSP-90 restored calcineurin activity to basal levels while higher concentrations (333 and 500 nM) increased calcineurin activity. In contrast, HSP-70 failed to activate calcineurin activity in the absence of calmodulin. Immunoprecipitation of HSP-90 from in vitro mixtures as well as protein extracts from LLCPK-1 cells demonstrates that calcineurin co-precipitates with HSP-90. In summary: (1) HSP-90 and 70 stimulate calcineurin V(max) in vitro; (2) non-specific protein interactions do not activate calcineurin activity; (3) HSP-70 and HSP-56 do not enhance HSP-90-induced activation of calcineurin; (4) HSP-70 and HSP-90 activate calcineurin via a calmodulin-dependent and independent pathways; (5) Calcineurin co-precipitates with HSP-90 from LLCPK-1 cells as well as cell-free in vitro preparations.
Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, 30322, USA.
J S
JS
Someren
L E
LE
Faber
J D
JD
Klein
J A
JA
Tumlin
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology; D019703:Calcineurin / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000502:physiology*; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D018840:HSP70 Heat-Shock Proteins / Q000494:pharmacology*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism / Q000494:pharmacology*; D020104:Immunophilins / Q000378:metabolism; D007700:Kinetics; D018374:LLC-PK1 Cells; D008970:Molecular Weight; D010446:Peptide Fragments / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding; D013552:Swine; D022021:Tacrolimus Binding Proteins; D013881:Thioridazine / Q000494:pharmacology
D065095:Calcineurin Inhibitors; D002147:Calmodulin; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D019703:Calcineurin; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins; D013881:Thioridazine