Interplay of Cullin5-HECTD3 ubiquitin ligases regulates stability of CRAF mutant associated with hypertrophy

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 CRAF ^D486N , 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.