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.
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
Howard
H
Doong
Kathryn
K
Rizzo
Shengyun
S
Fang
Vyta
V
Kulpa
Allan M
AM
Weissman
Elise C
EC
Kohn
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