The Interaction of Piasy with Trim32, an E3-Ubiquitin Ligase Mutated in Limb-girdle Muscular Dystrophy Type 2H, Promotes Piasy Degradation and Regulates UVB-induced Keratinocyte Apoptosis through NF B

Department of Dermatology and Program in Cell and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 10/2006; 281(35):25850-66. DOI: 10.1074/jbc.M601655200
Source: PubMed


Protein inhibitors of activated STATs (PIAS) family members are ubiquitin-protein isopeptide ligase-small ubiquitin-like modifier ligases for diverse transcription factors. However, the regulation of PIAS protein activity in cells is poorly understood. Previously, we reported that expression of Trim32, a RING domain ubiquitin-protein isopeptide ligase-ubiquitin ligase mutated in human limb-girdle muscular dystrophy type 2H (LGMD2H) and Bardet-Biedl syndrome, is elevated during mouse skin carcinogenesis, protecting keratinocytes from apoptosis induced by UVB and tumor necrosis factor-alpha (TNFalpha). Here we report that Trim32 interacts with Piasy and promotes Piasy ubiquitination and degradation. Ubiquitination of Piasy by Trim32 could be reproduced in vitro using purified components. Their interaction was induced by treatment with UVB/TNFalpha and involved redistribution of Piasy from the nucleus to the cytoplasm, where it accumulated in cytoplasmic granules that colocalized with Trim32. Piasy destabilization and ubiquitination required an intact RING domain in Trim32. The LGMD2H-associated missense point mutation prevented Trim32 binding to Piasy, and human Piasy failed to colocalize with human Trim32 in fibroblasts isolated from an LGMD2H patient. Trim32 expression increased the transcriptional activity of NFkappaB in epidermal keratinocytes, both under basal treatment and after UVB/TNFalpha treatment. Conversely, Piasy inhibited NFkappaB activity under the same conditions and sensitized keratinocytes to apoptosis induced by TNFalpha and UVB. Our results indicate that, by controlling Piasy stability, Trim32 regulates UVB-induced keratinocyte apoptosis through induction of NFkappaB and suggests loss of function of Trim32 in LGMD2H.

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    • "There is evidence that a number of the mammalian SUMO E3 ligases are targeted for ubiquitin-dependent proteasomal degradation. For example, PIAS1 is ubiquitinated by human homologs of seven in absentia 2 (hSIAh2) (Depaux et al. 2007), while PIASy is targeted for degradation by the ubiquitin ligase Trim32 (a protein mutated in limb-girdle muscular dystrophy type 2H and in Bardet–Biedl syndrome, and which is also elevated in mouse skin tumours induced in vivo by UVB) (Albor et al. 2006). Treatment of cells with MG132 promoted the interaction of TRIM32 and PIASy, resulting in the colocalisation of both proteins due to the redistribution of PIASy from the nucleus to the cytoplasm. "
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    • "Since TRIM32 can ubiquitinate malin substrates, we decided to investigate whether malin could ubiquitinate TRIM32 substrates. It has been described that TRIM32 ubiquitinates, among other substrates, dysbindin (a protein involved in endosomal-lysosomal trafficking and the genetic aetiology of schizophrenia) [33], and PIASy [Protein Inhibitor of Activated STAT (Signal Transducer and Activator of Transcription) isoform y, an E3-SUMO ligase] [34]. Following a similar approach as described above but using myc-dysbindin and myc-PIASy as substrates, we observed that only wild type TRIM32 but not the laforin-malin complex was able to ubiquitinate myc-dysbindin (Figure 8A) and myc-PIASy (Figure 8B). "
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    • "The sections were then incubated with affinity-purified chicken anti-Trim32 antibody generated in our laboratory (Albor et al., 2006) or goat anti-CCL20 antibody, clone AF360 (R&D Systems Inc., Minneapolis, MN) overnight at 4°C. The specificity of chicken anti-Trim32 antibody was verified by immunoblotting and immunostaining as described (Albor et al., 2006) and in the Supplemental data herein (Figure S1). The staining signal was visualized by the ABC approach (Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin. "
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