TRIM16 Acts As an E3 Ubiquitin Ligase and Can Heterodimerize with Other TRIM Family Members
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TRIM16 Acts as an E3 Ubiquitin Ligase and Can Heterodimerize with Other TRIM Family Members Jessica L. Bell1., Alena Malyukova1., Jessica K. Holien2, Jessica Koach1, Michael W. Parker2,3, Maria Kavallaris1, Glenn M. Marshall1,4*, Belamy B. Cheung1* 1 Children’s Cancer Institute Australia for Medical Research, Lowy Cancer Research Centre, University of New South Wales, Randwick, New South Wales, Australia, 2 St Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia, 3 Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia, 4 Centre for Children’s Cancer and Blood Disorders, Sydney Children’s Hospital, Randwick, New South Wales, Australia Abstract The TRIM family of proteins is distinguished by its tripartite motif (TRIM). Typically, TRIM proteins contain a RING finger domain, one or two B-box domains, a coiled-coil domain and the more variable C-terminal domains. TRIM16 does not have a RING domain but does harbour two B-box domains. Here we showed that TRIM16 homodimerized through its coiled-coil domain and heterodimerized with other TRIM family members; TRIM24, Promyelocytic leukaemia (PML) protein and Midline- 1 (MID1). Although, TRIM16 has no classic RING domain, three-dimensional modelling of TRIM16 suggested that its B-box domains adopts RING-like folds leading to the hypothesis that TRIM16 acts as an ubiquitin ligase. Consistent with this hypothesis, we demonstrated that TRIM16, devoid of a classical RING domain had auto-polyubiquitination activity and acted as an E3 ubiquitin ligase in vivo and in vitro assays. Thus via its unique structure, TRIM16 possesses both heterodimerization function with other TRIM proteins and also has E3 ubiquitin ligase activity. Citation: Bell JL, Malyukova A, Holien JK, Koach J, Parker MW, et al. (2012) TRIM16 Acts as an E3 Ubiquitin Ligase and Can Heterodimerize with Other TRIM Family Members. PLoS ONE 7(5): e37470. doi:10.1371/journal.pone.0037470 Editor: Beata G. Vertessy, Institute of Enzymology of the Hungarian Academy of Science, Hungary Received September 14, 2011; Accepted April 21, 2012; Published May 21, 2012 Copyright: ß 2012 Bell et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by an Early Career Development Fellowship, Cancer Institute NSW (B.C.); National Health & Medical Research Council (NHMRC) and a Biomedical PhD Scholarship (J.B.). M.W.P. is an Australian Research Council Federation Fellow and an NHMRC Honorary Fellow and thanks the Victorian State Government Operational Infrastructure Support Program for support. M.K. is an NHMRC Senior Research Fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (GMM); [email protected] (BBC) . These authors contributed equally to this work. Introduction a common ancestor [6,7]. TRIM16 does not have a RING domain but does harbour two B-box domains, leading to the The tripartite motif (TRIM)/Ring finger, B box, coiled-coil hypotheses that TRIM16 has RING-like domains and functions as (RBCC) family of proteins are defined by the presence of the an ubiquitin ligase. TRIM16’s C-terminus contains an RFP-like or tripartite motif. The TRIM protein family is composed of more B30.2/SPRY (B30.2) domain. than 70 highly conserved proteins which have been implicated in Several members of the TRIM protein family are known to a diverse range of biological processes including development, cell homodimerize and heterodomerize with other TRIMs, and the growth, differentiation, innate immune functions and cancer [1,2]. coiled-coil domain is generally required for these functional One poorly characterized member of this family is TRIM16. associations [8,9]. In this study, we show that TRIM16 can TRIM16 or estrogen-responsive B box protein (EBBP) is a positive homodimerize through its coiled-coil domain but also bind to transcriptional regulator of the retinoic acid receptor b (RARb ) 2 2 other TRIM proteins; Midline-1 (MID-1/TRIM18), Promyelocy- gene [3,4]. TRIM16 overexpression also enhances retinoid- tic leukemia protein (PML/TRIM19) and TRIM24 (TIF-1a). induced differentiation, reduces neuroblastoma cell growth, Additionally, our molecular modelling of TRIM16’s B-boxes migration, and significantly reduces tumorigenicity in vivo [5]. suggests zinc binding capability and we present evidence that B- Typically, TRIM proteins contain a Really Interesting New box domains have ubiquitination capability, leading to the Gene (RING) finger domain, one or two B-box domains, a coiled- suggestion that other B-box-containing proteins may perform coil domain and the more variable C-terminal domains. The a similar function. RING and B-box domains are both cysteine-rich and bind zinc atoms, indicating possible interactions with proteins, DNA and RNA. The RING domain has E3 ubiquitin-ligase potential and Results and Discussion can transfer ubiquitin to RING proteins as well as heterologous TRIM16 Homodimerizes via Its Coiled-Coil Domain substrates [1,2]. However, little is known about the function of B- In the large and evolutionary conserved TRIM family, specific box domains, although they occur widely in nature. Recently, the domains tend to operate as functional units. Many TRIM proteins first solved structure of a B-box domain displays a structure similar homodimerize via their coiled-coil domains [2,8,9]. To further to a RING domain, suggesting these domains evolved from PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e37470 TRIM16 Has Ubiquitin Ligase Activity Figure 1. TRIM16 homodimerizes through its coiled-coil domain. (A) TRIM16-GFP domain deletion plasmids. (B) Co-transfection of TRIM16- GFP and TRIM16-myc-His in HEK293 cells and subsequent immunoprecipitation by anti-myc antibody (Ab) and Western blot with anti-GFP antibody. Whole cell extract (WCE) used as total input. (C) TRIM16 homodimerizes through its coiled-coil domain. GFP deletion mutants were co-transfected with the TRIM16-myc-His vector. Anti-GFP antibody was used to pull down proteins binding the GFP tagged proteins and the TRIM16-myc-His was used to detect self-association via its different tag (right panel). Transfection efficiency was confirmed (left panel). TRIM16-GFP mutants were efficiently pulled down (middle panel). * non-specific bands, # refer to text. doi:10.1371/journal.pone.0037470.g001 investigate the function of TRIM16, we tested whether TRIM16 is vector pCMV6-AC-GFP and TRIM16-GFP or empty vector (EV) able to homodimerize. Two differently tagged TRIM16 expression pCDNA-myc-His and TRIM16-myc-His. Immunoprecipitation of vectors and 5 different deletion mutants of TRIM16 were used the myc-tag strongly indicated that TRIM16-GFP and TRIM16- (Figure 1A); TRIM16-GFP and TRIM16-myc-his with both tags myc-His were homodimerizing (Figure 1B). We confirmed these being fused separately to the C-terminus of the TRIM16 protein. results by using the anti-GFP antibody (Ab) for immunoprecipi- The full-length TRIM16 and 5 deletion constructs were used to tation, and investigating whether GFP-tagged deletion mutants of probe for the role of various domains in protein function TRIM16 were able to retain homodimerization with the (Figure 1A). HEK293 cells were co-transfected either empty TRIM16-myc-His. GFP fusion mutants bearing coiled-coil PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e37470 TRIM16 Has Ubiquitin Ligase Activity Figure 2. TRIM16 can heterodimerize with MID1, TRIM24 and PML. (A) Schematic structures of TRIM proteins used in heterodimerization studies. (B) TRIM16 binds MID1. Co-transfection of MID1-GFP and TRIM16-myc-His in HEK293 cells and subsequent immunoprecipitation by anti-myc antibody and Western blot with anti-GFP antibody. (C) MID1 was pulled down via its GFP antibody and a Western blot was performed to detect TRIM16-myc-His in the immunoprecipitated protein complex. (D) Whole cell lysates (WCL) of HEK293 cells transfected with empty vector (EV) or TRIM16-GFP were immunopreciptated with anti-GFP antibody. An anti-PML antibody was used to detect PML as a binding partner of TRIM16. (E) Lysates containing both TRIM16-GFP and TRIM24-His proteins were immunopreciptated with anti-GFP antibody. Anti-His antibody was used to detect the presence of TRIM24 in the TRIM16-associated complex. doi:10.1371/journal.pone.0037470.g002 PLoS ONE | www.plosone.org 3 May 2012 | Volume 7 | Issue 5 | e37470 TRIM16 Has Ubiquitin Ligase Activity domains (mutant 1, 3 and 5) were able to homodimerize with the particular substitution [6]. An unbiased blast search of the NCBI TRIM16-myc-His protein, indicating this region is required for database with TRIM16’s B-box1 sequence yields significant TRIM16 homodimerization (Figure 1C and 1A). However a weak sequence identity with human TRIM16, TRIM29, TRIM25, band (Lane 6 in Figure 1C) indicates that B-boxes are able to form MID1, RFP and MID2 (only TRIMs resulted, in order of some homodimers to a lesser extent. significance all with E-values ,0.009, and scores above 37.7). Analysis of TRIM16’s B-box2 sequences yielded similar results to TRIM16 Heterodimerizes with MID1, TRIM24 and PML B-Box1 (Figure 3B), although with a lower human sequence TRIM proteins have been shown to interact with other TRIM identity (28%) to MID1 (Sim, ExPASy, CA). When comparing the family members. For example, TRIM24 is known to bind PML full-length of the sequence, TRIM16 shares low sequence identity [10] and RET finger protein (RFP/TRIM27) through the B-box with other TRIMs such as TRIM29, TRIM25, RFP, MID1 and and coiled-coil domains [11].