14-3-3 proteins sequester a pool of soluble TRIM32 ubiquitin ligase to repress autoubiquitination and cytoplasmic body formation

Abstract

Deregulated expression of tripartite-motif protein 32 (TRIM32, an E3 ubiquitin-protein ligase) contributes to various diseases. Here we report, using quantitative proteomics and biochemistry, that 14-3-3 proteins bind to phosphorylated TRIM32 and prevent TRIM32 autoubiquitination and the formation of TRIM32-containing cytoplasmic bodies, potential autoregulatory mechanisms that can reduce the concentration of soluble free TRIM32. The 14-3-3–TRIM32 interaction was dependent on protein kinase A–catalyzed phosphorylation of TRIM32 at Ser651. We found that the inhibitory effect of 14-3-3 is, in part, a consequence of disrupting TRIM32's propensity to undergo higher order self-association without affecting its dimerization. Consequently, dimerized TRIM32 bound to 14-3-3 was sequestered in a distinct cytoplasmic pool away from the microtubule network, whereas a TRIM32 mutant that cannot bind 14-3-3 underwent multimerization and was unavailable to facilitate cell growth. Our results reveal a novel connection between ubiquitination and phosphorylation pathways, which could modulate a variety of cell events by stimulating the formation of the 14-3-3/TRIM32 signaling complex.