Degradation of heterotrimeric Galpha(o) subunits via the proteosome pathway is induced by the hsp90-specific compound geldanamycin.
One mechanism utilized by cells to maintain signaling pathways is to regulate the levels of specific signal transduction proteins. The compound geldanamycin (GA) specifically interacts with heat shock protein 90 (hsp90) complexes and has been widely utilized to study the role of hsp90 in modulating the function of signaling proteins. In this study, we used GA to demonstrate that levels of heterotrimeric Galpha subunits can be regulated through interactions with hsp90. In a dose-dependent manner, GA significantly reduced the steady state levels of endogenous Galpha(o) expression in two cell lines (PC12 and GH3) and had a similar effect on Galpha(o) transiently expressed in COS cells. Galpha(o) synthesis and degradation was studied in PC12 cells and in transiently transfected COS cells. (35)S labeling followed by immunoprecipitation demonstrated no effect of GA on the rate of Galpha(o) synthesis, but GA accelerated degradation of Galpha(o) in both PC12 cells and COS cells. The use of inhibitors, including lactacystin (a proteosome-specific inhibitor), suggests that Galpha(o) is predominantly degraded through the proteosome pathway. In vitro translated (35)S-labeled Galpha(o) could be detected in hsp90 immunoprecipitates, and this interaction did not require N-terminal myristoylation. Taken together, these results suggest that heterotrimeric Galpha(o) subunits are protected from degradation by interaction with hsp90 and that the interaction of Galpha subunits with heat shock proteins may be a general mechanism for regulating Galpha levels in the cell.
Busconi
L
L
Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA. lbusconi@hotmail.com
Guan
J
J
Denker
B M
BM
D000818:Animals; D016227:Benzoquinones; D019556:COS Cells; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D047029:Lactams, Macrocyclic; D009097:Multienzyme Complexes / Q000378:metabolism*; D016716:PC12 Cells; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011809:Quinones / Q000378:metabolism*; D051381:Rats; D013997:Time Factors; D014162:Transfection
D016227:Benzoquinones; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D009097:Multienzyme Complexes; D011809:Quinones; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D019204:GTP-Binding Proteins; C001277:geldanamycin