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The cellular level of TRIM31, an RBCC protein overexpressed in gastric cancer, is regulated by multiple mechanisms including the ubiquitin–proteasome system
Discovery Research Laboratory, Tokyo RD Center, Daiichi Pharmaceutical Co., Ltd, DaiichiSankyo group, 1613, Kitakasai 1Chome, Edogawaku, Tokyo 1348630, Japan
TRIM (tripartite motif) family proteins, comprising RING finger, B-box and coiled-coil domains, are involved in various cellular processes including tumour development and antiviral response. One of the family proteins, TRIM31, was originally identified as a gene induced by growth-suppressive retinoid. Our previous study showed that TRIM31 is up-regulated in stomach cancer and that TRIM31 protein possesses the common features of the TRIM protein family, for example, ubiquitin ligase activity and homo-oligomerization tendency. Interestingly, TRIM31 negatively regulates growth of certain cell types despite its overexpression in gastric cancer tissues. We herein demonstrated that upon exogenous expression in 293 cells, TRIM31 is polyubiquitylated, which promotes its degradation in the proteasome pathway. The proteasome-mediated degradation of endogenous TRIM31 was further confirmed in AsPC-1 pancreatic cancer cells. Thus, this posttranslational modification governs the intracellular abundance of TRIM31, which is also dependent on inducible transcription as well as alternative splicing. The complicated control of the intracellular TRIM31 protein level may relate to its seemingly contradictory behaviours in the cancer pathology or the urgent response to viral infection.
Key words: antivirus, MG132, proteasome, RING finger, TRIM31, ubiquitin
Abbreviations: IFN, interferon, TRIM, tripartite motif, RBCC, RING, B box and coiled coil, UPS, ubiquitin–proteasome system
To whom correspondence should be addressed (email firstname.lastname@example.org).
TRIM31 was identified first as a gene induced by the treatment of MCF-7 breast carcinoma cells with growth-suppressing retinoid (Dokmanovic et al., 2002). TRIM31 protein belongs to a subfamily of RING finger domain-containing proteins called the TRIM or RBCC (RING, B-box and coiled-coil) family. TRIM/RBCC proteins are associated with several pathological conditions such as immunological and developmental disorders, tumourigenesis and retroviral protective process (Meroni and Diez-Roux, 2005).
Through a genome-wide search for genes overexpressed in cancer (Sugiura et al., 2004), we recently found that TRIM31 expression is enhanced from the chronic gastritis phase or the early stage of stomach tumour generation (Sugiura and Miyamoto, 2008), where both apoptosis and proliferation are dysregulated (Jones et al., 1997). Furthermore, among normal adult tissues, TRIM31 transcription is limited to stomach, colon and small intestine that have a high proportion of dividing epithelial cells (Sugiura and Miyamoto, 2008). This observation has been validated by another group as high expression of TRIM31 protein was detected in normal gastrointestinal tracts including the stomach, small intestine and large intestine (Watanabe et al., 2009). From these results, TRIM31 is expected to contribute to the cell growth promotion; nevertheless, TRIM31 is inducible with growth-inhibitory retinoid and, consistently, has the ability to inhibit colony formation of HCT116 cells (Sugiura and Miyamoto, 2008) and to suppress Src-induced anchorage-independent growth of NIH3T3 cells (Watanabe et al., 2009).
These apparently inconsistent findings prompted us to presume that the cellular abundance of the RING finger-containing TRIM31 protein is finely tuned to adjust its biological function to the diverse contexts. In view of the protein modification pertinent to its turnover, RING finger proteins constitute a family that serves as E3 ubiquitin ligase (Lorick et al., 1999), and indeed, our biochemical characterization proved that TRIM31 displays in vitro autoubiquitylating activity as well (Sugiura and Miyamoto, 2008). Polyubiquitylation targets proteins for destruction by a multisubunit, ATP-dependent protease termed the proteasome and several RING finger proteins including TRIM proteins self-ubiquitylate in vivo, which leads to their own cellular degradation (Fang et al., 2000; Diaz-Griffero et al., 2006; Duan et al., 2008). Particularly, TRIM5 lends itself to restriction of virus infection by a mechanism involving its own proteasome-dependent degradation (Campbell et al., 2008). Since TRIM31 also exerts antiviral effects (Uchil et al., 2008), it is conceivable that the autoubiquitylating activity of TRIM31 regulates its own turnover as one of the mechanisms for strict controlling of its cellular amount. Therefore, in the present study, we sought to examine the relationship of TRIM31 with UPS (ubiquitin–proteasome system).
2. Materials and methods
All the experiments were repeated at least twice to confirm the reproducibility.
2.1. Cell culture and transfection
293 and AsPC-1 pancreatic cancer cells were cultured as described previously (Sugiura and Miyamoto, 2008). All the plasmid constructs were described by Sugiura and Miyamoto (2008). Expression vectors were transfected into the cells with the aid of LipofectAMINE plus (Invitrogen) as per the manufacturer (Sugiura et al., 2008a).
293 cells cultured in 10-cm plates were transfected with 4 μg pEF1/V5HisB–TRIM31 or pCI-neo3′FLAG–TRIM31. After 2 days, the cells were harvested and lysed in RIPA buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% NP40 (Nonidet P40), 0.05% SDS and 0.25% deoxycholate). After centrifuge, the supernatants were reacted with anti-V5 or anti-FLAG antibody agarose beads (Sigma) at 4°C overnight. Afterwards the beads were washed three times with TBS (Tris-buffered saline; 50 mM Tris/HCl, pH 7.5, 150 mM NaCl) containing 1% Triton X-100 and once with TBS. The proteins adsorbed onto the beads were solubilized in sample buffer containing 10% 2-mercaptethanol and subjected to 4–20% SDS/PAGE, followed by the Western blotting using anti-TRIM31 antibody at the dilution of 1:800 (Sugiura et al., 2008b).
2.3. Ubiquitylation of TRIM31
293 cells in six-well plates were transfected with 1 μg of the appropriate vectors, which were pCI-3′FLAG, pCI–5′HA, TRIM–FLAG and HA (haemagglutinin)–ubiquitin (Sugiura et al., 2007; Sugiura and Miyamoto, 2008), as shown (Figure 1C). Forty-eight hours later, lysates were prepared in RIPA buffer and immunoprecipitated with anti-FLAG antibody beads as above. The immunoprecipitates were heated in sample buffer under the reduced conditions, followed by 4–20% PAGE. After the immunoblot was conducted with anti-HA antibody (1:2000; Sigma), the membrane was reprobed with anti-TRIM31 antibody.
2.4. Proteasomal degradation
293 cells in six-well plates were transfected with 1 μg of the indicated plasmid. At 24-h posttransfection, the cells were treated with MG132 (Calbiochem) at 2.5 μM and cultured for an additional 24 h (Sugiura et al., 2007). Then, cell lysates were prepared as described above and analysed by the anti-TRIM31 Western blot. Likewise, AsPC-1 cells cultured in six-well dishes were treated with MG132 for 24 h and tested for TRIM31 expression as for the transfected 293 cells. The membranes of the blots were stripped and reprobed with anti-β-actin antibody (1:4000; Sigma). The signal from the β-actin probe was employed as a loading control. Densitometric analysis of the bands was performed with NIH image analyser (Scion Corporation).
3. Results and discussion
3.1. TRIM31 is ubiquitylated in vivo
In the previous study, we observed slowly migrating ladder bands when the lysates of 293 cells expressing FLAG-tagged TRIM31 from 35-mm culture was immunoblotted utilizing anti-TRIM31 antibody (Sugiura and Miyamoto, 2008). For more definite data, we intended to subject a greater amount of TRIM31 to the Western blot. 293 cells cultured in 10-cm plates were transfected with C-terminally FLAG-tagged TRIM31 expression vector, and the lysates were immunoprecipitated with anti-FLAG antibody. As can be seen, the immunoblot of the immunoprecipitates with anti-TRIM31 antibody revealed distinct ladder bands representing multimer forms and smear bands indicative of ubiquitylated proteins (Lorick et al., 1999) (Figure 1A). The similar experiments were carried out with the other vector for the expression of V5-tagged TRIM31 (Figure 1B). Again, we observed the typical smear bands along with faint ladders after probing the immunoprecipitates with anti-TRIM31 antibody. Consequently, the appearance of the high molecular weight smear or ladder bands can be ascribed to the intrinsic molecular nature of TRIM31 rather than the property of the tag, suggesting that TRIM31 is ubiquitylated in vivo.
We were still concerned about the tendency of TRIM proteins to oligomerize, which might result in the aggregated form with appreciable smear or ladder bands. To remove the possibility, we subsequently made an attempt to directly corroborate the ubiquitylation of TRIM31 by co-expressing HA-tagged ubiquitin. We transfected 293 cells cultured in six-well plates with plasmids for TRIM31–FLAG and HA–ubiquitin expression, as indicated (Figure 1C). The lysates of the transfected cells were immunoprecipitated with anti-FLAG antibody, and the immunoprecipitates were initially probed with anti-HA antibody, followed by reprobing with anti-TRIM31 antibody. The results showed that only when 293 cells were transfected with both TRIM31–FLAG and HA–ubiquitin vectors were the high molecular weight smear bands seen by the anti-HA immunoblot for the immunoprecipitate recovered with anti-FLAG antibody beads, ensuring that exogenous TRIM31 undergoes ubiquitylation in 293 cells.
3.2. TRIM31 is degraded in a proteasome-dependent pathway
Polyubiquitylated proteins are destined for degradation in a proteasome system (Hassink et al., 2005). In fact, several TRIM family members are degraded in the ubiquitin-dependent pathway (Diaz-Griffero et al., 2006; Duan et al., 2008). Accordingly, we were interested in whether the intracellular TRIM31 level would be similarly controlled via the proteasomal degradation pathway. To address the issue, we transfected 293 cells with a plasmid with no insert, intact TRIM31 sequence or its mutated sequence, which were afterwards treated with MG132, a proteasome inhibitor (Jensen et al., 1995). In mutated TRIM31, Cys31 and His33 in the RING finger domain were substituted with serine and glutamic acid, respectively, which compromised the in vitro autoubiquitylating activity of TRIM31 (Sugiura and Miyamoto, 2008). The anti-TRIM31 immunoblot analysis of the lysates illustrates that MG132 treatment increased the total amount of wild-type TRIM31 including clear ladder and degraded molecule bands (compare lanes 2 and 5 of Figure 2A). Likewise, an increase in the entire protein species was observable for the mutated form of TRIM31 deficient in E3 ligase activity (compare lanes 3 and 6 of Figure 2A). Hence, despite its self-ubiquitylation activity in vitro (Sugiura and Miyamoto, 2008), the in vivo ubiquitylation-dependent degradation of TRIM31 is likely to occur through a different E3 ligase(s). In this experiment, the steady-state level of mutant TRIM31 was lower than that of innate TRIM31 presumably because, apart from the regulated ubiquitylation, mutant TRIM31, destabilized by its inability to coordinate zinc atoms (Borden, 2000), is degraded in the proteasome-independent pathway, as in the case of mutated ZAP70 (Matsuda et al., 1999).
All the above experiments investigated the property of ectopic TRIM31 expressed in 293 cells. To elucidate the proteasomal degradation under the physiological conditions, we analysed AsPC-1 pancreatic cancer cells producing endogenous TRIM31 at a detectable level (Sugiura and Miyamoto, 2008) in terms of its turnover. Pancreatic AsPC-1 cells were chosen for this experiment since we failed to find a suitable gastric cancer cell line with an elevated TRIM31 expression and besides gastric cancers, pancreatic cancer tissues up-regulated TRIM31, implicating the physiological significance of its expression in AsPC-1 cells (Sugiura and Miyamoto, 2008). MG132-treated AsPC-1 cells were processed in the same way as the transfected 293 cells. We again observed that MG132 treatment resulted in the 2.1-fold increase in the amount of endogenous TRIM31 relative to the control culture (Figure 2B). Moreover, we previously detected ladder bands in the immunoblot of the AsPC-1 cell lysate employing anti-TRIM31 antibody (Sugiura and Miyamoto, 2008). Taken together, it can be concluded that the fate of TRIM31 is physiologically dictated by the ubiquitin–proteasome pathway. In strong support of this conclusion, a recent study on the global stability profiling of proteins by using the new technology presented the comprehensive list of proteins involved in the proteasome-mediated degradation pathway, in which TRIM31 is registered as a protein of the proteasome target (Yen et al., 2008). Thus, our results and those of Yen et al. (2008) complementarily reinforce the notion that TRIM31 is degraded in the proteasomal pathway.
3.3. Implications of the UPS for the function of TRIM31
The degradable property of TRIM31 might be exploited by the stomach cancer development, where its potential role is to keep the subtle balance between the proliferation and apoptosis of the susceptible cells (Jones et al., 1997; Sugiura and Miyamoto, 2008). Indeed, the expression of TRIM31 is increased in gastric cancer; nonetheless, it is inhibitory for cell growth (Sugiura and Miyamoto, 2008; Watanabe et al., 2009). The seeming disparity of TRIM31 might be, at least partially, reconciled by the fine tuning of its abundance through the degradable nature. One of the functions of TRIM proteins is interference with viral replication (Nisole et al., 2005; Ozato et al., 2008). It is recently reported that TRIM31 also has the ability to inhibit viral replication (Uchil et al., 2008). Degradation in the proteasome-dependent system is shared by other TRIM proteins competent to inhibit virus infection, as compiled (Table 1). Although the list is not exhaustive at present, and the data on the TRIM proteins in the degradation pathway are still lacking, we notice some tendency that the responsiveness to IFN (interferon) of antiviral TRIM proteins correlates with their involvement in the proteasomal degradation pathway. It appears that the expression of TRIM proteins with antiviral activity, in general, occurs in response to IFN (Carthagena et al., 2009), but in reality, some of them are not IFN-inducible (Table 1). It is possible that among the antiviral TRIM proteins, only those inducible by IFN require the mechanism underlying the management of their amount on demand.
Table 1 Involvement of human TRIM proteins with antiviral activity in the ubiquitin–proteasome system
bTarget viruses are derived from Nisole et al. (2005) and Carthagena et al. (2009). N-MLV, N-tropic murine leukaemia virus; HIV-1, human immunodeficiency virus type 1; ALV, avian leukaemia virus; EIAV, equine infectious anaemia virus. cReferences regarding the evidence for the involvement of the individual TRIM protein in the UPS are indicated. dNot documented. eHerpes simplex virus type 1, Ebola virus, lymphocytic choriomeningitis virus, Lassa virus, influenza virus, vesicular stomatitis virus, Rabies virus, HIV-1 and human foamy virus.
bTarget viruses are derived from Nisole et al. (2005) and Carthagena et al. (2009). N-MLV, N-tropic murine leukaemia virus; HIV-1, human immunodeficiency virus type 1; ALV, avian leukaemia virus; EIAV, equine infectious anaemia virus.
cReferences regarding the evidence for the involvement of the individual TRIM protein in the UPS are indicated.
eHerpes simplex virus type 1, Ebola virus, lymphocytic choriomeningitis virus, Lassa virus, influenza virus, vesicular stomatitis virus, Rabies virus, HIV-1 and human foamy virus.
This study has revealed that the complex regulation of the TRIM31 expression level is exerted at multiple levels. First, TRIM31 can be induced by various substances such as retinoid and IFNs (Dokmanovic et al., 2002; Carthagena et al., 2009). Following transcription, the alternative splicing of TRIM31 mRNA yields at least three isoforms with different expression patterns and possible distinct biological roles (Sugiura and Miyamoto, 2008), producing an additional level of complexity in the regulation of the TRIM31 function. Finally, as demonstrated in this study, TRIM31 degradation takes place through the ubiquitin–proteasome pathway.
Takeyuki Sugiura designed the research, performed the research, analysed the data and wrote the paper.
This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
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Received 30 October 2010/1 December 2010; accepted 13 January 2011
Published as Cell Biology International Immediate Publication 13 January 2011, doi:10.1042/CBI20100772
© The Author(s) Journal compilation © 2011 Portland Press Limited
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)