Proteostatic imbalance caused by CRAF (Raf1) kinase mutation alters MAPK signaling and contributes to RASopathies. CRAF proteostasis is maintained by chaperones, Hsp90 which aids in folding, maturation, and activation; whereas Hsp70 recruits ubiquitin ligase CHIP for proteasomal degradation. However, the proteostasis of disease-associated CRAF mutants remains undefined. Here, we identify HECTD3, a HECT-ubiquitin ligase in promoting degradation of hypertrophic CRAF mutants. Interestingly, increased Hsp90 binding to CRAF plays a determinant factor in degradation via HECTD3. Transient increase in Hsp90-CRAF association upon Hsp90 inhibition promote ubiquitination, whereas prolonged inhibition stabilizes mutant CRAF, indicating temporally distinct chaperone functions. Notably, HECTD3 undergoes proteosomal degradation under stress via Cullin-RING ubiquitin ligase Cul5 in a neddylation-dependent manner. Overexpression of Cul5 thus reduces HECTD3 level resulted in stabilization of CRAFD486N, while promoting degradation of wild-type CRAF. Therefore, modulation of the Cul5-HECTD3 axis alters hypertrophic signaling in cardiomyocyte cells. These results establish a hierarchical proteostasis mechanism in which Hsp90 binding primes substrate for HECTD3-mediated ubiquitination, and Cul5-controlled ligase turnover collectively determine mutant CRAF fate.
Roy, S., Mukherjee, S., Mandal, A. K.
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