Studies of Hsp90 function on signal transduction molecules in C2C12 cells and effects of novobiocin on Hsp90 conformation and function and mechanism of regulation of heme-regulated eIF2alpha kinase by gas molecules

Abstract

Scope and Method of Study: The purpose of this study was to investigate: the function of Hsp90 on signal transduction pathways required for C2C12 myoblast differentiation; to characterize the effect of the Hsp90-inhibitor novobiocin on the structure and function of Hsp90; and to elucidate the mechanism of regulation of heme-regulated eIF2alpha kinase (HRI) by the gas molecules, nitric oxide (NO) and carbon monoxide (CO). Hsp90-dependent signaling pathways were examined in differentiating C2C12 myoblasts, using the pharmacological agents geldanamycin, novobiocin, and okadaic acid, pulse-chase labeling, immunoadsorption of protein kinases, and western blotting. The mechanism of inhibition of Hsp90 by novobiocin was analyzed using pull-down assays and western blotting, proteolytic fingerprinting, and protein kinase assays. Regulation of HRI by NO and CO was studied using coupled transcription/translation of site-specific and deletion mutants of HRI in nuclease treated reticulocyte lysate, followed by pull-down and kinase assays to analyze interdomain interactions and the activity of HRI.

Findings and Contributions: Inhibition of Hsp90 function induced apoptosis in differentiating C2C12 myoblasts by depleting ErbB2, Akt(PKB) and Fyn kinases, which modulate signal transduction pathways critical for cell differentiation and survival, and blocking myogenin expression, which is required for transcriptional activation of muscle specific genes. Hsp90 modulated Akt activity via protein phosphatase 2A. Novobiocin bound to the C-terminal region of Hsp90, induced specific conformational changes in Hsp90's structure, altered Hsp90's interaction with its co-chaperone partners, and inhibited the maturation of HRI. These results verify that novobiocin is an Hsp90 inhibitor. The interaction of the N-terminal heme-binding domain (NT-HBD) of HRI with the catalytic domain of HRI was disrupted by NO, but enhanced by CO or heme. A deletion mutant HRI was hyperactivated in heme-deficient lysate, identifying a region within the NT-HBD of HRI that is critical for the regulation of HRI activity and modulating inter-domain interactions.