CAIR-1/BAG-3 abrogates heat shock protein-70 chaperone complex-mediated protein degradation: accumulation of poly-ubiquitinated Hsp90 client proteins.
BAG family proteins are regulatory co-chaperones for heat shock protein (Hsp) 70. Hsp70 facilitates the removal of injured proteins by ubiquitin-mediated proteasomal degradation. This process can be driven by geldanamycin, an irreversible blocker of Hsp90. We hypothesize that CAIR-1/BAG-3 inhibits Hsp-mediated proteasomal degradation. Human breast cancer cells were engineered to overexpress either full-length CAIR-1 (FL), which binds Hsp70, or a BAG domain-deletion mutant (dBAG) that cannot bind Hsp70. FL overexpression prevented geldanamycin-mediated loss of total and phospho-Akt and other Hsp client proteins. dBAG provided no protection, indicating a requirement for Hsp70 binding. Ubiquitinated Akt accumulated in FL-expressing cells, mimicking the effect of lactacystin proteasomal inhibition, indicating that CAIR-1 inhibits proteasomal degradation distal to protein ubiquitination in a BAG domain-dependent manner. Protein protection in FL cells was generalizable to downstream Akt targets, GSK3beta, P70S6 kinase, CREB, and other Hsp client proteins, including Raf-1, cyclin-dependent kinase 4, and epidermal growth factor receptor. These findings suggest that Hsp70 is a chaperone driving a multiprotein degradation complex and that the inhibitory co-chaperone CAIR-1 functions distal to client ubiquitination. Furthermore, poly-ubiquitination is not sufficient for efficient proteasomal targeting of Hsp client proteins.
Doong
Howard
H
Molecular Signaling Section, Laboratory of Pathology and Regulation of Protein Function Laboratory, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA. ek1b@nih.gov
Rizzo
Kathryn
K
Fang
Shengyun
S
Kulpa
Vyta
V
Weissman
Allan M
AM
Kohn
Elise C
EC
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D048868:Adaptor Proteins, Signal Transducing; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D016227:Benzoquinones; D001943:Breast Neoplasms; D002352:Carrier Proteins / Q000235:genetics / Q000494:pharmacology*; D003546:Cysteine Endopeptidases; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D015870:Gene Expression; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D007163:Immunosorbent Techniques; D047029:Lactams, Macrocyclic; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D046988:Proteasome Endopeptidase Complex; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011809:Quinones / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured; D025801:Ubiquitin / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C414431:BAG3 protein, human; D016227:Benzoquinones; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D009097:Multienzyme Complexes; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011809:Quinones; D025801:Ubiquitin; C067713:lactacystin; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine; C001277:geldanamycin