TRIM32 Protein Sensitizes Cells to Tumor Necrosis Factor (TNFα)-induced Apoptosis via Its RING Domain-dependent E3 Ligase Activity against X-linked Inhibitor of Apoptosis (XIAP)

Abstract

TRIM32, which belongs to the tripartite motif (TRIM) protein family, has the RING finger, B-box, and coiled-coil domain structures common to this protein family, along with an additional NHL domain at the C terminus. TRIM32 reportedly functions as an E3 ligase for actin, a protein inhibitor of activated STAT y (PIASy), dysbindin, and c-Myc, and it has been associated with diseases such as muscular dystrophy and epithelial carcinogenesis. Here, we identify a new substrate of TRIM32 and propose a mechanism through which TRIM32 might regulate apoptosis. Our overexpression and knockdown experiments demonstrate that TRIM32 sensitizes cells to TNF␣-induced apoptosis. The RING domain is necessary for this pro-apoptotic function of TRM32 as well as being responsible for its E3 ligase activity. TRIM32 colocalizes and directly interacts with X-linked inhibitor of apoptosis (XIAP), a well known cancer therapeutic target, through its coiled-coil and NHL domains. TRIM32 overexpression enhances XIAP ubiquitination and subsequent proteasomemediated degradation, whereas TRIM32 knockdown has the opposite effect, indicating that XIAP is a substrate of TRIM32. In vitro reconstitution assay reveals that XIAP is directly ubiquitinated by TRIM32. Our novel results collectively suggest that TRIM32 sensitizes TNF␣-induced apoptosis by antagonizing XIAP, an anti-apoptotic downstream effector of TNF␣ signaling. This function may be associated with TRIM32-mediated tumor suppressive mechanism.TRIM32/HT2A is a member of the tripartite motif (TRIM) 5 protein family. To date, 72 TRIM-encoding genes have been identified in the human genome, all sharing the same overall arrangement of RING, B-box, and coiled-coil domains. Their main divergences are in the number of B-boxes and the natures of their C-terminal domains, with most TRIMs possessing one or two additional C-terminal domains, such as B30.2-like/RFP/ SPRY, NHL, ARF, PHD, and BROMO domains (1). TRIM32 has one B-box and six repeats of the NHL motif (2).Several TRIM genes have been associated with disease. For example, mutations in TRIM18/MID1, TRIM20/PYRIN, and TRIM37/MUL have been associated with X-linked Optiz G/BBB syndrome (3), familial Mediterranean fever (4), and mulibrey nanism (5), respectively. Mutations in TRIM32 were recently linked to limb girdle muscular dystrophy type 2H and sarcotubular myopathy, both of which may be caused by the same mutation (D487N) at the third NHL repeat (6). Three additional mutations in the first (R394H), fourth (T520fsX13), and fifth (D588del) NHL repeats of TRIM32 have also been found to cause limb girdle muscular dystrophy type 2H (7). Several TRIM genes have been associated with tumorigenesis; for example, TRIM19/PML (8), TRIM24/TIF1␣ (9, 10), TRIM25/EFP (11), and TRIM27/RFP (12) have been linked to tumor initiation and progression, and the level of TRIM32 mRNA was found to be up-regulated in epidermal cancers (13). However, the precise biological functions of the majority of TRIM family members have not yet been characterized.The RING domain is ...

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

“…Consistent with previous findings (Albor et al, 2006;Locke et al, 2009;Ryu et al, 2011), the expressed FLAG-TRIM32 was biologically active and was heavily autoubiquitylated in the cells, as demonstrated by a smear of high-molecular-mass species of HA-ubiquitin (Fig. 4A, lanes 2 and 8).…”

“…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 other TRIM32 constructs (Albor et al, 2006;Locke et al, 2009;Ryu et al, 2011), the FLAG-tagged TRIM32 we generated spontaneously formed CBs when transiently expressed in cultured cells (Fig. 3A, left panel).…”

Satellite cell senescence underlies myopathy in a mouse model of limb-girdle muscular dystrophy 2H

“…TRIM32 has been shown to be elevated in epidermal and epithelial tumorigenic cells, where its antiapoptotic and carcinogenic roles have been discovered (19,38,39). However, TRIM32 overexpression/knockdown experiments in established cell culture lines have suggested a proapoptotic, tumor suppressive role for TRIM32 (40). Our data demonstrate that apoptosis is not perturbed in TRIM32-deficient skeletal muscle and that it is not an essential pathogenic feature of Trim32 -/-myopathy.…”

“…Apoptosis contributes to skeletal muscle remodeling to maintain a constant nuclearto-cytoplasm ratio during hind limb suspension or immobilization (37). Moreover, it's been previously shown that TRIM32 may play an antiapoptotic role in epidermal keratinocytes (38) and squamous epithelial cells (39) but a proapoptotic role in some established cell culture lines (40). Therefore, we examined whether apoptosis is altered in skeletal muscle during hind limb suspension/reloading in the absence of TRIM32.…”