E3 ubiquitin ligase Cullin-5 modulates multiple molecular and cellular responses to heat shock protein 90 inhibition in human cancer cells.
The molecular chaperone heat shock protein 90 (HSP90) is required for the activity and stability of its client proteins. Pharmacologic inhibition of HSP90 leads to the ubiquitin-mediated degradation of clients, particularly activated or mutant oncogenic protein kinases. Client ubiquitination occurs via the action of one or more E3 ubiquitin ligases. We sought to identify the role of Cullin-RING family E3 ubiquitin ligases in the cellular response to HSP90 inhibition. Through a focused siRNA screen of 28 Cullin-RING ligase family members, we found that CUL5 and RBX2 were required for degradation of several HSP90 clients upon treatment of human cancer cells with the clinical HSP90 inhibitor 17-AAG. Surprisingly, silencing Cullin-5 (CUL5) also delayed the earlier loss of HSP90 client protein activity at the same time as delaying cochaperone dissociation from inhibited HSP90-client complexes. Expression of a dominant-negative CUL5 showed that NEDD8 conjugation of CUL5 is required for client degradation but not for loss of client activity or recruitment of clients and HSP90 to CUL5. Silencing CUL5 reduced cellular sensitivity to three distinct HSP90 inhibitors, across four cancer types driven by different protein kinases. Our results reveal the importance of CUL5 in multiple aspects of the cellular response to HSP90 inhibition.
Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London SM2 5NG, UK.
Rahul S
RS
Samant
Paul A
PA
Clarke
Paul
P
Workman
D016227:Benzoquinones / Q000494:pharmacology; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D044842:Cullin Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D000075687:NEDD8 Protein; D009369:Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D057170:Translational Research, Biomedical; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism
D016227:Benzoquinones; D044842:Cullin Proteins; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D000075687:NEDD8 Protein; D034741:RNA, Small Interfering; D018719:Erb-b2 Receptor Tyrosine Kinases; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins; C496871:CUL5 protein, human; C082860:NEDD8 protein, human; C112765:tanespimycin; C118307:RNF7 protein, human; C508053:ERBB2 protein, human
BRAF; ERBB2; oncoprotein; protein degradation; protein quality control