TRIM8 modulates STAT3 activity through negative regulation of PIAS3

Fumihiko Okumura 2 Yui Matsunaga 2 Yuta Katayama 0 1 Keiichi I. Nakayama 0 1 Shigetsugu Hatakeyama 2 0 CREST, Japan Science and Technology Agency (JST) , Kawaguchi, Saitama 332-0012 , Japan 1 Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University , Fukuoka, Fukuoka 812-8582 , Japan 2 Department of Biochemistry, Hokkaido University Graduate School of Medicine , N15, W7, Kita-ku, Sapporo, Hokkaido 060-8638 , Japan - Summary TRIM8 is a member of the protein family defined by the presence of a common domain structure composed of a tripartite motif: a RING-finger, one or two B-box domains and a coiled-coil motif. Here, we show that TRIM8 interacts with protein inhibitor of activated STAT3 (PIAS3), which inhibits IL-6-dependent activation of STAT3. Ectopic expression of TRIM8 cancels the negative effect of PIAS3 on STAT3, either by degradation of PIAS3 through the ubiquitin-proteasome pathway or exclusion of PIAS3 from the nucleus. Furthermore, expression of TRIM8 in NIH3T3 cells enhances Src-dependent tumorigenesis. These findings indicate that TRIM8 enhances the STAT3-dependent signal pathway by inhibiting the function of PIAS3. e c n e ic Introduction lleS iTnhteheubeilqimuiitnina-timonedoiaftsehdoprtr-olitveeodlyrteicguplaatthowryayprhoatesinans (iPmepteorrst,an1t99ro8l)e, C including those that contribute to the cell cycle, cellular signaling fo in response to environmental stress or extracellular ligands, la morphogenesis, secretion, DNA repair and organelle biogenesis rnu (aHttaecrshhmkeonatnodf Cuibeicqhuaintionvetor, 1th9e98ta).rgTehtepsryosteteinm croesnpsoisntssibolfe sfeovretrhael Jo components that act in concert (Hershko and Ciechanover, 1992; Scheffner et al., 1995), including a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2) and a ubiquitin-protein isopeptide ligase (E3). E3 is thought to be the component of the ubiquitin conjugation system that is most directly responsible for substrate recognition (Scheffner et al., 1995). On the basis of structural similarity, E3 enzymes have been classified into three families: the HECT (homologous to E6-AP COOH terminus) family (Hershko and Ciechanover, 1998; Huibregtse et al., 1995), the RING-finger-containing protein family (Freemont, 2000; Joazeiro and Weissman, 2000; Lorick et al., 1999) and the U-box family (Aravind and Koonin, 2000; Cyr et al., 2002; Hatakeyama et al., 2001). The superfamily of tripartite-motif-containing (TRIM) proteins is defined by the presence of a tripartite motif composed of a RING domain, one or two B-box motifs and a coiled-coil region (the so-called RBCC motif) (Meroni and Diez-Roux, 2005; Nisole et al., 2005). Many TRIM proteins are induced by type I and type II interferons (IFNs), suggesting that TRIM proteins have an important role in anti-viral and anti-microbial systems (Rajsbaum et al., 2008). The human TRIM8 gene is expressed in a variety of tumors, including anaplastic oligodendroglioma, and maps to chromosome 10q24.3, a region that shows frequent deletion or loss of heterozygosity in glioblastomas (Vincent et al., 2000). Therefore, TRIM8 is also designated glioblastoma-expressed RING-finger protein (GERP). It has been reported that TRIM8 localizes to specific nuclear bodies and cytosolic speckles in U2OS and HeLa cells (Reymond et al., 2001). TRIM8 has also been shown by yeast two-hybrid screening to be a suppressor of cytokine signaling (SOCS)-1 interacting protein (Toniato et al., 2002). TRIM8 mRNA can be induced by IFNg in murine B lymphoid M12 cells, murine fibroblasts and HeLa cells and the N-terminal 204 amino acids of TRIM8 accelerate the degradation of SOCS-1 and reverse SOCS1-mediated inhibition of JAK-STAT activation by IFNg (Toniato et al., 2002). However, it is not clear whether full-length TRIM8 truly regulates the JAK-STAT pathway. Protein inhibitor of activated STAT3 (PIAS3) has been reported to inhibit the DNA-binding activity of signal transducer and activator of transcription 3 (STAT3), followed by the suppression of STAT3-mediated gene activation (Chung et al., 1997). Since many cytokine receptors do not have intrinsic tyrosine-kinase activity, ligand engagement leads to the activation of receptorassociated tyrosine kinases, which are usually members of the Janus kinase (JAK) family (Darnell, 1998; Heinrich et al., 2003; Stark et al., 1998; Yu et al., 2007). STAT3 is also activated by growth-factor receptors, including epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR) and vascular endothelial growth factor receptor (VEGFR) (Yu et al., 2007). Furthermore, many tumor-produced factors, such as IL-10, IL-6 and VEGF, which are crucial for both tumor growth and immunosuppression, activate STAT3 to create an efficient feed-forward mechanism to ensure increased STAT3 activity both in tumor cells and in tumorassociated immune cells (Yu et al., 2007). STAT3 is constitutively activated in cells transformed by the oncoprotein Src, which is a non-receptor tyrosine kinase (Yu et al., 1995). Since inhibition of STAT3 signal blocks the transformation of fibroblasts by Src, STAT3 has been shown to be an important molecule for oncogenesis by Src (Bromberg et al., 1998; Turkson et al., 1998). However, a direct role of STAT3 in oncogenesis was shown using a constitutively active STAT3 mutant, which transforms fibroblasts in culture and allows the transformed cells to form tumors in mice (Bromberg et al., 1999). Under physiological conditions in normal cells, the activation of STAT proteins is rapid and transient because they are negatively regulated by proteins such as SOCS and PIAS (Alexander, 2002; Kubo et al., 2003; Shuai and Liu, 2005). In this study, we performed yeast two-hybrid screening using TRIM8 as bait and found that PIAS3 is a TRIM8-interacting protein. TRIM8 negatively regulates PIAS3 by degradation through the ubiquitin-proteasome pathway and/or exclusion of PIAS3 from the nucleus. These findings show that expression of TRIM8 might cause prolonged activity of STAT3, and thereby could induce oncogenesis. Results TRIM8 interacts with PIAS3 Yeast two-hybrid screening was performed to identify proteins that interact with TRIM8. A mouse T-cell cDNA library was screened using TRIM8 lacking a RING domain (DRING) as a bait, because immunoblot analysis showed that full-length TRIM8 was only faintly expressed in yeast cells. From 5 105 transformants that were able to grow on Leu- and Trp-deficient medium, seven positive clones were isolated after two rounds of growth in the absence of His and screening for b-galactosidase activity. The ce nucleotide sequence of one of these seven clones led to the n identification of PIAS3. As shown in Fig. 1A, TRIM8 has a RING e ic domain, two B-boxes and a coiled-coil domain. PIAS3 also has a S RING domain (MIZ-Zn finger) as well as a SAP box, a PINIT lle domain, and an acidic domain (D (...truncated)