TRIM32 is an E3 ubiquitin ligase for dysbindin

Abstract

Mutations in the gene encoding tripartite motif protein 32 (TRIM32) cause two seemingly diverse diseases: limb-girdle muscular dystrophy type 2H (LGMD2H) or sarcotubular myopathy (STM) and Bardet–Biedl syndrome type 11(BBS11). Although TRIM32 is involved in protein ubiquitination, its substrates and the molecular consequences of disease-causing mutations are poorly understood. In this paper, we show that TRIM32 is a widely expressed ubiquitin ligase that is localized to the Z-line in skeletal muscle. Using the yeast two-hybrid system, we found that TRIM32 binds and ubiquitinates dysbindin, a protein implicated in the genetic aetiology of schizophrenia, augmenting its degradation. Small-interfering RNA-mediated knock-down of TRIM32 in myoblasts resulted in elevated levels of dysbindin. Importantly, the LGMD2H/STM-associated TRIM32 mutations, D487N and R394H impair ubiquitin ligase activity towards dysbindin and were mislocalized in heterologous cells. These mutants were able to self-associate and also co-immunoprecipitated with wild-type TRIM32 in transfected cells. Furthermore, the D487N mutant could bind to both dysbindin and its E2 enzyme but was defective in monoubiquitination. In contrast, the BBS11 mutant P130S did not show any biochemical differences compared with the wild-type protein. Our data identify TRIM32 as a regulator of dysbindin and demonstrate that the LGMD2H/STM mutations may impair substrate ubiquitination.

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

“…The interaction of all these 14-3-3 isoforms with FLAG-TRIM32 were stimulated by coexpression of cPKA in HEK293 cells (Fig. 2C), a cell line widely used in the study of TRIM32 interaction and localization (Kano et al, 2008;Locke et al, 2009;Ryu et al, 2011). Phosphate incorporation into TRIM32 under these conditions was confirmed using phospho-PKA substrate antibodies (Fig.…”

“…Like many TRIMs, TRIM32 has two potential autoregulatory properties. One is that it often self-associates and forms insoluble high-molecular-mass puncta called cytoplasmic bodies (CBs) when transiently expressed in cells (Albor et al, 2006;Locke et al, 2009). Although the role of CBs in TRIM32 functions is not fully understood, recent studies, particularly with TRIM19 and TRIM5, have suggested that CBs may function as aggresome precursors or storage compartments that maintain the proper level of soluble free TRIM proteins (Song et al, 2005;Diaz-Griffero et al, 2006) or exchange the proteins between CBs and a more diffuse cytoplasmic fraction (Campbell et al, 2007).…”

E3 ubiquitin ligase TRIM32 negatively regulates tumor suppressor p53 to promote tumorigenesis

“…13,14 TRIM32 ubiquitinates dysbindin that may contribute to TRIM32's role in skeletal muscle and neuronal cell differentiation. 17 Most recently, p73 was reported to upregulate the basal levels of TRIM32, and furthermore, TRIM32 ubiquitinates p73 in neuronal cells, which might contribute to TRIM32's function in neuronal development and differentiation. 41 The regulation of TRIM32 by p53 and p73 appears to be different and highly context dependent.…”

“…TRIM32 ubiquitinates dysbindin that may contribute to TRIM32's role in skeletal muscle and neuronal cell differentiation. 17 TRIM32 ubiquitinates NF-kB inhibitor Piasy and tumor suppressor Abi2, which may contribute to TRIM32's role in tumorigenesis. 16,18 In this study, we identified TRIM32 as a novel p53 target and negative regulator for p53.…”

“…TRIM32 was reported to possess the E3 Ub ligase activity. 16,17,18 These findings raised an interesting question that whether TRIM32 is an E3 Ub ligase for p53. To test this hypothesis, p53-null human lung H1299 cells were co-transfected with vectors expressing WT p53 and TRIM32.…”