The TRIM-FLMN Protein TRIM45 Directly Interacts with RACK1 and Negatively Regulates PKC-Mediated Signaling Pathway

ORIGINAL ARTICLE The TRIM-FLMN protein TRIM45 directly interacts with RACK1 and negatively regulates PKC-mediated signaling pathway

T Sato1,2, H Takahashi2, S Hatakeyama2, A Iguchi1 and T Ariga1

The receptor for activated C-kinase (RACK1), a scaffolding protein that participates in the protein kinase C (PKC) signaling pathway, has an important role in shuttling active PKCs to its substrate. Indeed, recent studies have revealed that RACK1 has an important role in tumorigenesis and that enhancement of the feed-forward mechanism of the c-Jun N-terminal kinase (JNK)–Jun pathway via RACK1 is associated with constitutive activation of MEK (MAPK-ERK kinase)–ERK (extracellular signal-regulated kinase) signaling in human melanoma cells. Taken together, RACK1 additionally has a very important role in the mitogen-activated protein kinase (MAPK) signaling pathway. Here, we show that one of the tripartite motif-containing (TRIM) family ubiquitin ligases, TRIM45, is a novel RACK1-interacting protein and downregulates MAPK signal transduction. Importantly, the expression of TRIM45 is induced when growth-promoting extracellular stimuli activate the MAPK signaling pathway, resulting in attenuation of activation of the MAPK pathway. These ﬁndings suggest that TRIM45 functions as a member of the negative feedback loop of the MAPK pathway.

Oncogene (2015) 34, 1280–1291; doi:10.1038/onc.2014.68; published online 31 March 2014

INTRODUCTION The receptor for activated C-kinase (RACK1), a scaffolding protein A variety of extracellular stimuli induce intracellular signal that participates in the protein kinase C (PKC) signaling pathway, is transduction including those that contribute to cell proliferation, a member of the tryptophan-aspartate repeat (WD-repeat) family of cell cycle progression, cell differentiation and apoptosis in proteins.13 RACK1 was originally identified as a scaffolding protein eukaryotic cells. Intracellular signal transduction, often referred for activated PKCβII, not a substrate for a specific enzyme, and it to as mitogen-activated protein kinase (MAPK) pathways, is was revealed that activation of several PKC isoforms including generated by activation of cell surface receptors, which, in turn, PKCβII leads to interaction with RACK1 and then RACK1 shuttles the successively changes enzymatic activity of proteins in the active kinase to the site of its substrate.13–17 As PKC has been cytoplasm and nucleus.1–5 The MAPK cascade is composed of implicated in cell proliferation, carcinogenesis and cell cycle three kinds of protein kinase: MAPK kinase kinase (MAPKKK), progression by means of rewired ERK signal transduction, RACK1, MAPK kinase (MAPKK) and MAPK.6 The previous studies revealed as a preferable partner of the active form of PKC, could potentially that there are three distinct and parallel MAPK pathways regulate cellular functions through the PKC signaling pathway. categorized by their functions: c-Jun N-terminal kinase (JNK), Indeed, several studies have revealed that RACK1 has a pivotal role p38 and extracellular signal-regulated kinase (ERK).7–9 A number in carcinogenesis and that its expression level is altered in several of studies have revealed that serum growth factors and phorbol human carcinomas.18–21 Moreover, RACK1 is not only a scaffolding esters such as phorbol-12-myristate-13-acetate (PMA), can protein for PKC but also serves as an adaptor protein for other enhance cell growth and survival through activation of the promiscuous signaling enzymes including the cyclic adenosine MAPK-ERK kinase (MEK) 1/2–ERK1/2 pathway. On the other hand, monophosphate-specific phosphodiesterase PDE4D5.22 As RACK1 it has been shown that hyperactivation of the Ras–Raf–MEK–ERK has been reported to interact with one of the MAPK cascade pathway causes cellular carcinogenesis.10 In contrast, the p38 and signaling molecules, JNK, RACK1 may serve as a scaffolding protein JNK pathways are regulated by various environmental stresses for JNK signaling.23 It has also been shown that phosphorylation of such as arsenite (type 1 stress) and genotoxic agents (type 2 JNK by PKC occurs on Ser129 and requires the existence of RACK1 stress).11 Some of these stresses can induce programmed cell and that inhibition of RACK1 expression attenuates JNK activation. death via Stress-activated protein kinase pathways, which have a Inhibition of RACK1 expression induces to sensitize ultraviolet- central role in control of immunoresponse and inflammation. The induced apoptosis in human melanoma cells and takes away the mechanism of the mainstream of each of these MAPK pathways tumorigenesis.23,24 In addition, it has been reported that enhance- has been clarified to a certain extent. In addition, the mechanism ment of the feed-forward mechanism of the JNK–Jun pathway via that links ERK with JNK signaling in a certain human cell line has RACK1 is associated with constitutive activation of MEK–ERK been reported.12 However, the precise mechanism that mediates signaling in human melanoma cells.12 Results of those previous between the Raf–MEK–ERK cascade and Stress-activated protein studies indicate that RACK1 has a very important role in MAPK kinase cascade remains unknown. signaling pathways.

1Department of Pediatrics, Hokkaido University Graduate School of Medicine, Sapporo, Japan and 2Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Japan. Correspondence: Dr T Sato, Department of Pediatrics, Hokkaido University Graduate School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo, Hokkaido 060-8638, Japan. E-mail: [email protected] Received 19 April 2013; revised 8 January 2014; accepted 12 January 2014; published online 31 March 2014 TRIM45 regulates activation of RACK1/PKC pathway T Sato et al 1281

Flag-TRIM45: IP HA-RACK1: Mr (k) IP: HA Flag-TRIM45 WCL 57 Mr (k) IB:Flag 57 TRIM45 IP: Flag 36.5 HA-RACK1 IB: TRIM45 36.5 IB:HA RACK1 57 Flag-TRIM45 IB: RACK1 10% input IB:Flag

36.5 HA-RACK1 IB:HA

Flag-TRIM45: Flag-TRIM45: HA-RACK1: GST-RACK1: PMA (5nM): GST: Mr (k) GST-RACK1 36.5 HA-RACK1 IB: GST IP: Flag IB:HA IP: Flag Flag-TRIM45 57 IB:Flag IB: Flag Flag-TRIM45 Flag-TRIM45 57 GST-RACK1 IP: HA IB:Flag HA-RACK1 36.5 input IB: GST GST IB:HA HA-RACK1 36.5 10% input IB:HA Flag-TRIM45 IB: Flag Flag-TRIM45 57 IB:Flag

HA-RACK1 Flag-TRIM45 Merge RACK1 Flag-TRIM45 Merge

Arsenite Arsenite (0.5mM) (0.5mM)

Figure 1. Interaction between RACK1 and TRIM45. (a) In vivo binding assay between RACK1 and TRIM45. Expression vectors encoding HA-tagged RACK1 and Flag-tagged TRIM45 were transfected into HEK293T cells. (b) Interaction between endogenous RACK1 and TRIM45 in HeLa cells. Whole-cell lysates were immunoprecipitated with anti-RACK1 antibody or mock (control IgG) and immunoblotted with an antibody to TRIM45 or RACK1. (c) In vitro pull-down assay between RACK1 and TRIM45. Recombinant GST-tagged RACK1 and His6-Flag-TRIM45 were mixed in combination as indicated. The reaction mixtures were immunoprecipitated with M2-agarose and then immunoblotted with anti-GST antibody. (d) In vivo binding assay between RACK1 and TRIM45 with or without PMA. HEK293T cells were transfected with plasmids encoding HA-tagged RACK1 and Flag-tagged TRIM45. Forty hours after transfection, the cells were incubated with or without PMA (5 nM) for 6 h. After PMA stimulation, cell lysates were immunoprecipitated with anti-HA or anti-Flag antibody and immunoblotted with anti-Flag and anti-HA antibodies. (e) Colocalization of Flag-TRIM45 (red) with HA-RACK1 (green, left panels) or endogenous RACK1 (green, right panels), which is recruited into stress granules, after arsenite (0.5 mM) stimulation in COS-7 cells. Scale bars: 10 μm. Data are representative of three or more independent experiments.

The superfamily of tripartite motif-containing (TRIM) proteins immunoglobulin (IG-FLMN) domain in its carboxy-terminal region. are characterized by the presence of a RING finger, one or two It has been reported that TRIM45 has some a role as a zinc-binding motifs called B-boxes, an associated coiled-coil downregulator in the MAPK signaling pathway through activator region and unique carboxyl-terminal domains.25–27 TRIM family protein-1 (AP-1)/E twenty-six-like transcription factor 1 (Elk-1).34 proteins are involved in a wide range of biological processes, and However, the detailed molecular function of TRIM45 has not been their varieties often cause diverse pathological conditions such as elucidated. developmental disorders, neurodegenerative diseases and Here, we show that TRIM45 is a novel RACK1-interacting protein carcinogenesis.28–33 TRIM45, a member of the TRIM family, and downregulates MAPK signal transduction through inhibiting contains a RING finger domain, two B-boxes, and a coiled-coil RACK1/PKC complex formation. Interestingly, the expression of region in its amino-terminal region. TRIM proteins can be classified TRIM45 is induced upon activation of a MAPK signaling pathway into subfamilies on the basis of differences in their carboxy- by growth-promoting extracellular stimuli, resulting in attenuation terminal domain structure. In addition to its common motifs of activation of the MAPK pathway. These findings suggest of the TRIM family proteins, TRIM45 contains a filamin-type that TRIM45 functions as a factor responsible for negative

GST-RACK1: WD1 WD2 WD3 WD4 WD5 WD6 WD7 Flag-TRIM45: RACK1 GST-RACK1 derivatives IP: Flag R B BBC FLMN TRIM45 (WT) IB: GST Flag-TRIM45 IB: Flag TRIM45 (ΔRING) TRIM45 (ΔB-box) GST-RACK1 derivatives TRIM45 (ΔBBC) input GST TRIM45 (FLMN) IB: GST Flag-TRIM45 IB: Flag

GST-RACK1: Flag-TRIM45: Flag-TRIM45: Pseudo RACK1 peptide: HA-RACK1: r (k) 36.5 HA-RACK1 GST-RACK1 IB: HA Flag-TRIM45 WT 57 IP: Flag IP: Flag 43 Flag-TRIM45 ΔB-box 36.5 Flag-TRIM45 FLMN IB: GST 28 IB: Flag Flag-TRIM45 36.5 HA-RACK1 IB: Flag IB: HA Flag-TRIM45 WT 57 GST-RACK1 10% input 43 Flag-TRIM45 ΔB-box 36.5 input GST 28 Flag-TRIM45 FLMN IB: Flag IB: GST Flag-TRIM45 IB: Flag Figure 2. The domains of RACK1 critical for interaction with PKC are also required for binding to TRIM45. (a) Schematic structure of RACK1. GST-tagged recombinant deletion mutants of RACK1 in which one or more WD40 repeats are deleted were used in (b). (b) In vitro pull-down assay between deletion mutants of RACK1 and TRIM45. Recombinant GST-tagged full-length RACK1 (WT) or GST-tagged deletion mutants of RACK1 and Flag-TRIM45 were mixed in combination as indicated. The reaction mixtures were immunoprecipitated with M2-agarose and then immunoblotted with anti-GST antibody. (c) Schematic representation of TRIM45 and deletion mutants. R, Ring-ﬁnger domain; B, B-box domain; BBC, B-box C-terminal domain; FLMN, Filamin-type immunoglobulin domain. Flag-tagged deletion mutants of TRIM45 were used in (d). (d) In vivo assay for binding between deletion mutants of TRIM45 and RACK1. HA-tagged RACK1 and Flag-tagged full-length (WT) or deletion mutants of TRIM45 were co-transfected into HEK293T cells. Immunoprecipitated Flag-TRIM45 (WT) and Flag-TRIM45 mutants were probed for co-precipitating HA-RACK1 (top panel). (e) The pseudo RACK1 peptide interferes with the interaction between RACK1 and TRIM45 in a dose-dependent manner in vitro. Recombinant GST-tagged RACK1, Flag-tagged TRIM45 recombinant proteins and the pseudo RACK1 peptide (10 or 30 μM) were mixed in combination as indicated. The reaction mixtures were immunoprecipitated with Flag-agarose and then immunoblotted with anti-GST or anti-Flag antibody. Data are representative of three or more independent experiments.

feedback of the MAPK pathway and is an antitumor molecule in proteins of glutathione S-transferase (GST)-tagged RACK1 and cancer cells. Flag-tagged TRIM45. Flag-tagged TRIM45 bound to M2-agarose was incubated with GST-RACK1, followed by washing the resin, RESULTS and immunoblot analysis was then performed with an anti-GST antibody. This analysis showed that RACK1 specifically associated RACK1 directly interacts with TRIM45 with TRIM45 in vitro (Figure 1c). Next, to address whether phorbol With the aim of identifying the TRIM45-interacting protein, we ester affects the binding of RACK1 to TRIM45, we performed performed several rounds of yeast two-hybrid screening. One of co-precipitation experiments again: exogenously expressed the positive clones had sequence identities with complementary Flag-TRIM45 co-precipitated with co-expressed HA-RACK1 with DNA encoding human RACK1 (Supplementary Figure 1). Inter- or without PMA stimulation (Figure 1d). The results suggest that action between RACK1 and TRIM45 was confirmed both in vivo PMA stimulation induces the expression of TRIM45 and promotes and in vitro by co-precipitation experiments: Flag-tagged TRIM45 interaction between RACK1 and TRIM45. Moreover, it has been (Flag-TRIM45) co-precipitated with co-expressed HA-tagged reported that RACK1 is sequestered into stress granules under RACK1 without any stimulation (Figure 1a). Co-precipitation of conditions of type 1 environmental stress.11 We performed endogenous RACK1 and endogenous TRIM45 was also demon- immunofluorescence staining to check the pattern of intracellular strated, indicating that RACK1 associates with TRIM45 in vivo TRIM45 protein translocation with arsenite. We obtained results (Figure 1b). Furthermore, to verify the direct interaction between showing that RACK1 was sequestered into stress granules. these proteins, we carried out in vitro pull-down assays. For Immunofluorescence staining showed that TRIM45 colocalized this purpose, we generated bacterially expressed recombinant with RACK1 with arsenite (Figure 1e).

Oncogene (2015) 1280 – 1291 © 2015 Macmillan Publishers Limited TRIM45 regulates activation of RACK1/PKC pathway T Sato et al 1283 Critical regions of RACK1 for PKC binding are also required for system, and we examined endogenous RACK1 expression by interaction with TRIM45 immunoblot analysis (Supplementary Figure 3d). Protein stability RACK1 is a member of the WD40 superfamily of proteins with analysis showed that exogenous TRIM45 expression does not seven WD40 repeats (Figure 2a). It has been shown that WD2, affect the stability of RACK1. WD3 and WD6 regions of RACK1 contribute to the PKCβII binding interface to maintain PKC in an active state by means of Interaction between RACK1 and PKCβII is inhibited by TRIM45 competition experiments with small peptide sequences derived It is known that activation of PKCβII leads to interaction between 35 fi from these domains. Thus, to further con rm the binding region RACK1 and the active form of the kinase, and RACK1 then shuttles of RACK1 to TRIM45, we performed in vitro binding assay using the active PKCβII to the site of its appropriate substrates. We GST-tagged recombinant proteins of several deletion mutants of previously observed that exogenous TRIM45 expression negatively RACK1 and compared their abilities to bind with Flag-tagged regulates nuclear factor-κB-mediated transcription and suppresses TRIM45 recombinant protein (Figure 2b). An in vitro binding assay cell proliferation. We speculated that TRIM45 acts as a repressor showed that a mutant with deletion of WD6 to WD7 domains between the region downstream of PKC pathways and the nuclear Δ – Δ ( WD6 7) and a mutant with deletion of the WD7 domain ( WD7) factor-κB and/or MAPK signaling pathways (Supplementary attenuated the binding to TRIM45. In addition, mutants with Figure 4a).36 This observation led us to hypothesize that TRIM45 Δ – deletion of WD1 to WD3 domains ( WD1 3) and deletion of WD5 can inhibit the function of RACK1 by inhibiting interaction Δ – to WD7 domains ( WD5 7) abolished the binding to TRIM45, between RACK1 and PKCβII directly, not through the ubiquitin– indicating that the WD1 to WD3 region and WD5 are essential for proteosomal degradation system. Initially, we performed a interaction with TRIM45 with RACK1 and that the WD2, WD3 and co-precipitation experiment. However, interaction between β WD6 regions of RACK1, putative PKC II-binding sites, may also be Flag-TRIM45 and HA-PKCβII was not observed (Supplementary needed for binding to TRIM45. Conversely, to determine which Figure 4b). Next, the interaction between RACK1 and PKCβII was region of TRIM45 is required for the interaction with RACK1, in vivo examined in vivo by co-precipitation experiments: HA-PKCβII co-precipitation experiments were performed using TRIM45 co-precipitated with co-expressed Flag-tagged RACK1 without any deletion mutants (Figures 2c and 2d). An in vivo binding assay stimulation (Figure 3a). Cells were transfected with different showed that Flag-TRIM45 (FLMN) more weakly interacted with amounts of Flag-TRIM45. We confirmed that the expression levels Δ HA-RACK1 than did TRIM45 (wild type (WT)) or TRIM45 ( B-box). of endogenous RACK1 and PKC were not affected by TRIM45 or fi These ndings suggest that the B-box C-terminal domain (BBC empty vector transfection (Supplementary Figure 4c). Immunoblot domain) of TRIM45 is important for interaction with RACK1. To analysis showed that exogenously expressed TRIM45 attenuated know the characteristics of TRIM45 in more detail, we tested the interaction between RACK1 and PKCβII in a TRIM45 dose- whether TRIM45 (WT) and a series of truncate mutants have the dependent manner. Similarly, under the condition of PMA ability to form homodimers (Supplementary Figure 2). In vivo stimulation in HeLa cells, co-precipitation of endogenous RACK1 co-precipitation experiments using Flag-tagged full-length and endogenous PKC could be more clearly demonstrated TRIM45, its deletion mutants and HA-tagged full-length TRIM45 without exogenously expressed TRIM45 (Figure 3b). The same showed that deletion mutant of TRIM45 having BBC domain can tendency for the level of immunoprecipitates derived from interact with another full-length TRIM45. Moreover, to clarify exogenously expressed Flag-RACK1 and HA-PKCβII to be increased whether TRIM45 interacts with regions of RACK1 that are similar to without overexpressed TRIM45 under the condition of PMA PKC interaction sites, we carried out in vitro pull-down assays with stimulation was shown by co-precipitation assays in vivo GST-RACK1 protein, Flag-TRIM45 recombinant protein and pseudo (Figure 3c). Furthermore, to test whether TRIM45 directly inhibits RACK1 peptides that block the RACK1/PKC interaction. The in vitro the interaction between RACK1 and PKCβII, we carried out in vitro binding assay showed that dose escalation of introduced pseudo pull-down assays with GST-RACK1 protein, HA-tagged PKCβII RACK1 peptides attenuated the interaction between RACK1 and protein and Flag-TRIM45 recombinant protein. Consistent with the TRIM45 recombinant proteins, indicating that the pseudo RACK1 results of the binding assay in vivo, the in vitro binding assay peptides compete with RACK1 proteins and bind to TRIM45. showed that dose escalation of introduced TRIM45 recombinant Therefore, this observation suggests that TRIM45 binds to RACK1 protein attenuated the interaction between RACK1 and PKCβII through the sites that are necessary for activating PKC (Figure 2e). recombinant proteins, indicating that TRIM45 directly inhibited interaction of these proteins (Figure 3d). To test whether TRIM45 has a ubiquitin ligase activity exogenous expression of TRIM45 affects the kinase activity of PKC, we performed an in vitro kinase assay using myelin basic TRIM45 has a RING finger domain at its NH terminus and belongs 2 protein (MBP), which is one of the PKC substrates, and PKC to the TRIM family of proteins, which have been reported to be E3 immunoprecipitated from HeLa cells or HeLa cells stably expres- ubiquitin ligases. Initially, to verify whether TRIM45 actually sing Flag-tagged TRIM45. As shown in Figure 3e, MBP phosphor- mediates an E3 ligase activity, we performed in vitro ubiquitylation ylation by PKC immunoprecipitated from HeLa cells expressing assays with various combinations. For this purpose, we deter- Flag-tagged TRIM45 was much less than MBP phosphorylation by mined E2 preferences of TRIM45 (Supplementary Figure 3a). PKC immunoprecipitated from mock HeLa cells. Our finding Immunoblot analysis revealed that Flag-tagged TRIM45 ubiquity- suggested that exogenous expression of TRIM45 represses PKC lates itself in the presence of E1, E2 (Ubc2B), ubiquitin, adenosine activity in cells (Figure 3e, Supplementary Figures 4d and e). triphosphate and TRIM45 (Supplementary Figure 3c). However, although ubiquitylation activity was also exhibited in the presence of these components with GST-tagged RACK1 recombinant Exogenously overexpressed TRIM45 represses transcription of a protein, immunoblot analysis using an anti-RACK1 antibody series of genes induced by serum stimulation revealed that RACK1 itself was not ubiquitylated by TRIM45. It has been shown that the RACK1 gene promoter contains Consistent with these results, TRIM45 did not affect RACK1 a number of transcription factor binding sites including serum- ubiquitination in vivo (Supplementary Figure 3b). Next, to verify responsive elements such as a cognitive site for AP-1.12,37 whether TRIM45 participates in proteosomal degradation of Twelve-O-tetradecanoylphorbol-13-acetate-responsive element is RACK1, we examined protein stability of RACK1 in the presence recognized by AP-1 and is known to be at the terminus of a of an inhibitor of protein biosynthesis, cycloheximide. For this complicated pathway, including the PKC signaling pathway, analysis, we generated a HeLa S3 cell line in which Flag-tagged which transmits the effect of carcinogenic promoters to the TRIM45 (WT) is stably expressed by using a retroviral expression transcriptional machinery. Binding of a transcription factor to the

HA-PKCβII: Flag-RACK1: IP: Flag-TRIM45: Flag-TRIM45: Mr (k) Flag-RACK1 Mr (k) 36.5 IB: PKC PKC 1.00 0.78 0.05 :Fold 70 IB: Flag 1.0 3.0 :Fold IB: RACK1 RACK1 HA-PKCβII 28 70 IB: HA IB: Flag Flag-TRIM45 57 36.5 Flag-RACK1 IB: Flag 70 HA-PKCβII IB: HA GST-RACK1: β Flag-TRIM45 HA-PKC II: 57 IB: Flag Flag-TRIM45: Mr (k) 57 PMA (5nM): GST-RACK1 1.00 0.53 0.08 :Fold β HA-PKC II: IB: GST Flag-RACK1: β 70 HA-PKC II Flag-TRIM45: IB: HA Mr (k) Flag-RACK1 57 GST-RACK1 36.5 IB: Flag 43 HA-PKCβII 70 36.5 IB: HA GST Flag-RACK1 IB: GST 36.5 IB: Flag 70 Flag-TRIM45 HA-PKCβII 70 IB: Fag IB: HA HA-PKCβII Flag-TRIM45 70 57 IB: HA IB: Flag

Flag-TRIM45:

Autoradiography MBP IP: anti-PKC IB: anti-PKC PKC

IB: anti-PKC PKC

Input IB: anti-FLAG Flag-TRIM45

Figure 3. Interaction between RACK1 and PKCβII is impaired by TRIM45. (a) TRIM45 inhibits interaction between RACK1 and PKCβII in a dose- dependent manner. HEK293T cells were transfected with plasmids encoding HA-PKCβII, Flag-RACK1 and Flag-TRIM45 (5, 10 μg), followed by immunoprecipitation with anti-HA antibody. Immunoprecipitates were subjected to immunoblot analysis with anti-Flag-RACK1 or anti-HA antibody. (b) In vivo binding assay between endogenous RACK1 and endogenous PKC with or without exogenously expressed Flag-TRIM45 in HeLa cells. HeLa cells were transfected with expression plasmids encoding Flag-tagged TRIM45 or an empty vector. Forty hours after transfection, the cells were incubated with PMA (5 nM) for 6 h. After PMA stimulation, cell lysates were immunoprecipitated with anti-RACK1 antibody or mock (control IgG) and immunoblotted with an antibody to PKC, Flag or RACK1. The fold numbers were provided from this single blot. (c) TRIM45 also impairs interaction between RACK1 and PKCβII in a dose-dependent manner under the condition of PMA stimulation. HEK293T cells were transfected with plasmids encoding HA-PKCβII, Flag-RACK1 and Flag-TRIM45 (5, 10 μg). Forty hours after transfection, the cells were incubated with and without PMA (5 nM) for 6 h, followed by immunoprecipitation with anti-HA antibody. Immunoprecipitates were subjected to immunoblot analysis with anti-Flag-RACK1 or anti-HA antibody. (d) TRIM45 interferes with the interaction between RACK1 and PKCβII in a dose-dependent manner in vitro. Recombinant GST-tagged RACK1, His6-HA-PKCβII and His6-Flag-TRIM45 (0.5, 1 μg) were mixed in combination as indicated. The reaction mixtures were immunoprecipitated with HA-agarose and then immunoblotted with anti-GST, anti-HA or anti-Flag antibody. Data are representative of three or more independent experiments. (e) Exogenous expression of TRIM45 represses the activity of PKC. An in vitro kinase assay was performed using MBP and PKC immunoprecipitated from mock HeLa cells or HeLa cells stably expressing Flag-tagged TRIM45. MBP phosphorylation by PKC immunoprecipitated from HeLa cells expressing Flag-tagged TRIM45 was less than MBP phosphorylation by PKC immunoprecipitated from mock HeLa cells.

serum response element is critical for activating transcription of control cells for the indicated time after serum induction. Next, to certain genes in response to serum stimulation and, as a result, clarify whether deletion mutants of TRIM45 affect transcription of RACK1 expression is also known to be upregulated after serum target genes of RACK1, we performed real-time PCR to compare stimulation. We hypothesized that exogenously expressed TRIM45 c-Jun and Early growth response protein-1 (EGR1) mRNA levels suppresses transcription of certain genes, for instance, c-Jun and under the same conditions as the above. Transcription of c-Jun Fos, in response to serum stimulation. We carried out real-time and EGR1 genes was not affected by overexpression of TRIM45 PCR to compare mRNA levels of c-Jun, Fos and JunB as target (ΔBBC) and TRIM45 (FLMN), whereas it was negatively regulated genes of RACK1 under the condition of serum stimulation with by overexpression of TRIM45 (WT) and TRIM45 (ΔB-box) (Figures and without overexpressed TRIM45 in HeLa cells (Figure 4a). 4b and c). As TRIM45 (WT) and TRIM45 (ΔB-box), but not TRIM45 Notably, mRNA levels of all of these genes were negatively (FLMN), have the ability to interact with RACK1 and form a dimer regulated in TRIM45-overexpressed cells compared with those in with another TRIM45 (Supplementary Figure 2), it was speculated

c-Jun Fos JunB 12 35 18 ** * 16 10 30 * 14 25 8 12 20 10 6 15 8 4 6 10 4 2 5 2 0 0 0 Duration of 0015 15 0015 15 0015 15 serum induction (min)

c-Jun EGR1 25 300 * * 250 20 200 15 150 10 100

5 50

0 0 Duration of 0015 15 0 15 0015 15 0 15 serum induction (min)

c-Jun c-Jun 45 25 * 40 Flag-TRIM45: 35 20 30 15 25 IB: Flag 20 10 15 10 5 5 IB: β-actin 0 0 Duration of 0015 15 0 15 0015 15 0 15 serum induction (min)

** ** ** 1.2 * 1

0.8

0.6

0.4

0.2

0 Mock TRIM45

Figure 4. Exogenously expressed TRIM45 represses the induction of AP-1 genes after serum stimulation. (a) Quantification of the amounts of c-Jun, Fos and JunB by real-time quantitative PCR using HeLa cells that were transfected with a plasmid encoding TRIM45 or corresponding empty vector. For transient TRIM45 transfection, 36 h after transfection, cell cultures at 50–70% confluency were washed twice with phosphate- buffered saline, starved in serum-free medium for >12 h, and then treated with serum-containing medium for the times indicated in each figure. The relative mRNA expression level of each gene in cells that had been treated without serum induction was defined as 1. Data are means ± s.d. of values from three independent experiments. P-values for indicated comparisons were determined by Student’s t-test (*Po0.05; **Po0.01). (b) Exogenously expressed full-length TRIM45, but not TRIM45 (FLMN) and TRIM45 only BBC domain deletion mutant, causes downregulation of induction of c-Jun and EGR1 genes after serum stimulation. Using the same method as in (a), mRNA expression levels of c-Jun and EGR1 were measured by real-time quantitative PCR. Data are means ± s.d. of values from three independent experiments (*Po0.05). (c) Immunoblot analysis of FLAG-tagged TRIM45 (WT) and TRIM45 (ΔBBC). Expression vectors encoding FLAG-tagged TRIM45 (WT) and TRIM45 (ΔBBC) were transfected into HeLa cells. The cells were assayed for real-time quantitative PCR in (b). Anti-β-actin antibody was used as an internal control. (d) Exogenously expressed TRIM45 represses the expression of cell cycle-related genes. TRIM45 significantly downregulates transcription of cyclin A2, cyclin D1, cyclin D3 and cyclin E genes in HeLa cells. The relative mRNA expression level of each gene in cells that had been transfected with an encoding empty vector was defined as 1. Data are means ± s.d. of values from three independent experiments (*Po0.05; **Po0.01).

© 2015 Macmillan Publishers Limited Oncogene (2015) 1280 – 1291 TRIM45 regulates activation of RACK1/PKC pathway T Sato et al 1286 that (i) overexpressed TRIM45 inhibited RACK1 activity through its stably knocked down compared with the level in cells transfected direct interaction or that (ii) overexpressed TRIM45 dimerized with with sh-control (Figures 5d and e, Supplementary Figure 6). endogenous TRIM45 and inhibited RACK1 function indirectly. Our Moreover, we carried out real-time quantitative PCR using observation suggests that the BBC domain of TRIM45 is important sh-TRIM45 HeLa cell lines to compare mRNA levels of c-Jun and for interaction with RACK1 and is required for transcriptional EGR1 under the condition of serum stimulation with or without regulation of c-Jun and EGR1 activity after serum stimulation. As calphostin C (500 nM, 1 h), which is a specific PKC inhibitor. As negative control, transcription of Myc and brain-derived neuro- shown in Figure 5h, mRNA levels of both of the genes were trophic factor (BDNF) genes, which are also considered to be negatively regulated by the addition of calphostin C in TRIM45- regulated by RACK1 gene activity,38 did not change with or knocked down cells. These results suggest that TRIM45 negatively without TRIM45 (Supplementary Figure 5). In addition, it has been regulates the expression of c-Jun and EGR1 genes through a reported that certain isoforms of PKC regulate cell cycle PKC-dependent pathway. To verify whether the increase in AP-1 progression through activation of the cyclin promoter, although gene transcription induced by silencing of TRIM45 has an the precise mechanisms are elusive.39 Therefore, to test whether influence on cell growth, we performed a cell proliferation assay exogenously expressed TRIM45 affects transcription of cyclin using TRIM45 knockdown cell lines. The growth rate of HeLa cell family genes, we carried out real-time PCR to compare mRNA lines in which TRIM45 was knocked down was significantly levels of those genes with or without overexpressed TRIM45 in increased compared with that of cells transfected with the HeLa cells (Figure 4d). The mRNA levels of cyclin family genes corresponding empty vector (Figure 5f). These findings indicate were also downregulated in overexpressed TRIM45 cells com- that TRIM45 affects cell proliferation via regulation of transcription pared with those in control cells. of genes in response to serum stimulation. Next, we examined whether exogenous expression of TRIM45 could maintain the MAPK pathway under the condition of using forced expression of Knockdown of TRIM45 increases transcription of AP-1 genes HA-tagged PKC-ΔNPS as previously reported.39 PKC-ΔNPS con- induced by serum stimulation and promotes growth of HeLa cells structs encode mutants in which the N-terminal sequence from To clarify whether silencing of TRIM45 conversely causes elevation the N terminus through the pseudosubstrate sequence is deleted. of mRNA levels of these AP-1 genes and promotes cell Thus, PKC-ΔNPS works as a constitutive active mutant of PKC proliferation, we next performed real-time PCR to compare mRNA independent of binding with RACK1. As shown in Figure 5g, levels of c-Jun and Fos under the condition of serum stimulation immunoblot analysis showed that endogenous EGR1 levels were with or without RNA interference of TRIM45. To knock down elevated more in cells in which HA-tagged PKC-ΔNPS had been endogenous TRIM45, three different targeting small interfering transfected, whereas EGR1 protein was only faintly expressed in RNAs (siRNAs) specific for human TRIM45 (si-TRIM45 #1-#3) or non- cells in which HA-tagged PKC-WT had been transfected. Notably, targeting siRNA as a control (si-control) were transfected into HeLa the expression levels of EGR1 in cells in which PKC-ΔNPS and cells (Figure 5a). The mRNA levels of c-Jun and Fos were elevated Flag-TRIM45 had been transfected were elevated, whereas the in HeLa cells transfected with the si-TRIM45s compared with the expression levels of EGR1 in cells in which PKC-WT and levels in cells transfected with si-control (Figure 5b). We also Flag-TRIM45 had been transfected not elevated. These observa- examined the effect of TRIM45 knockdown on cell growth. For this tions suggest that TRIM45 can regulate the expression level of purpose, we generated HeLa cell lines in which TRIM45 is stably EGR1 under the condition with not PKC-ΔNPS, which has no knocked down. Three different targeting short hairpin RNAs region for binding to RACK1 but PKC-WT. (shRNA) specific for human TRIM45 (sh-TRIM45 #1-#3) or non- targeting shRNA as a control (sh-control) were introduced (Figure 5c). Consistent with the results obtained by using Silencing of TRIM45 increases the amplification loop of c-Jun si-TRIM45, transcription levels of c-Jun, Fos and EGR1 were activation via the RACK1/PKC/JNK/c-Jun feedback loop elevated in cells transfected with the sh-TRIM45s compared with The results shown in Figures 1d and 3c led us to predict that the the levels in cells transfected with sh-control, and the expression expression level of endogenous TRIM45 may be changed by some level of EGR1 was elevated in HeLa cell lines in which TRIM45 was extracellular stimuli that participate in cell proliferation via MAPK

Figure 5. Knockdown of TRIM45 enhances transcription of Ap-1 genes by serum stimulation. (a) Knockdown of TRIM45 in HeLa cells using siRNAs targeting human TRIM45. Expression levels of TRIM45 proteins derived from HeLa cells transfected with siRNA specific for human TRIM45 (si-TRIM45 #1-#3) or with non-targeting siRNA as a control (si-control) were measured by immunoblot analysis. Cell lysates were subjected to immunoblotting using anti-TRIM45 or anti-β-actin antibody. (b) Knockdown of TRIM45 increases the induction of c-Jun and Fos genes in HeLa cells after serum stimulation. For transient si-TRIM45s and si-control transfection, 36 h after transfection, cells were starved by culturing at 50–70% confluency in serum-free medium for >12 h and then treated with serum-containing medium for the times indicated. The relative mRNA expression level of each gene in cells that had been treated without serum induction was defined as 1. P-values for indicated comparisons were determined by Student’s t-test (**Po0.01). Data are means ± s.d. (c) Knockdown of TRIM45 in HeLa cells. ShRNAs targeting human TRIM45 (sh-TRIM45 #1-#3) or scrambled shRNA as a negative control (sh-control) were introduced into HeLa cells by a retroviral expression system. Cell lysates were subjected to immunoblotting using anti-TRIM45 or anti-β-actin antibody. (d) Knockdown of TRIM45 using shRNAs causes upregulation of c-Jun, Fos and EGR1 gene expression in HeLa cells. P-values for indicated comparisons were determined by Student’s t-test (*Po0.05). (e) Expression level of EGR1 was elevated by 1 h of serum stimulation after serum starvation in HeLa cell lines in which TRIM45 was stably knocked down compared with the level in cells transfected with sh-control. (f) Silencing of TRIM45 promotes HeLa cell proliferation. HeLa cells in which sh-TRIM45s and sh-control were introduced by a retroviral expression system and seeded at 1 × 105 cells in 60 mm dishes. The cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% (v/v) fetal bovine serum and then counted. The number of live cells was determined by counting using a hemocytometer after Trypan blue staining at the indicated times. Data shown are average values from three independent experiments. P-values for indicated comparisons were determined by Student’s t-test (*Po0.05; **Po0.01). Data are means ± s.d. (g) Exogenously expressed TRIM45 can regulate the expression level of EGR1 under the condition of overexpression of PKC-WT but not that of PKC-ΔNPS. Results of Immunoblot analysis of endogenous EGR1 in HeLa cells in which HA-tagged PKC-WT or HA-tagged PKC -ΔNPS had been transfected with or without Flag-tagged TRIM45 are shown. Anti-β-actin antibody was used as an internal control. Data are representative of three or more independent experiments. (h) Knockdown of TRIM45 using shRNAs causes downregulation of c-Jun and EGR1 gene expression with calphostin C in HeLa cells. Cells were starved by culturing at 50–70% confluency in a serum-free medium for >12 h and then treated with serum-containing medium for the times indicated with or without calphostin C (500 nM). The relative mRNA expression level of each gene in cells that had been treated without serum induction was defined as 1. P-values for indicated comparisons were determined by Student’s t-test (*Po0.05). Data are means ± s.d.

Oncogene (2015) 1280 – 1291 © 2015 Macmillan Publishers Limited TRIM45 regulates activation of RACK1/PKC pathway T Sato et al 1287 pathways. We performed real-time PCR to test whether TRIM45 JNK and that enhanced activity of JNK together with elevated mRNA levels change after treatment of HeLa cells with PMA expression of c-Jun is involved in maintaining continuous (Figure 6a). Following PMA and serum stimulation, transcription of activation of c-Jun in certain human carcinoma cell lines.12 Results the TRIM45 gene was elevated, and the expression level of TRIM45 showing that transcription of c-Jun and Fos was activated in HeLa was elevated in HeLa cells (Figure 6a). Recent studies have cells transfected with si-TRIM45 and sh-TRIM45 compared with the revealed that the RACK1–PKC complex causes phosphorylation of levels in control cells led us to speculate that TRIM45 has an

c-Jun Fos ** 40 ** ** 35 70 30 ** 60 25 ** 50 20 40 IB:TRIM45 TRIM45 15 30 10 20 IB: β-actin 5 10 0 0 0150015 15 0 15 Duration of 015 0015 15 0 15 serum induction (min)

c-Jun Fos 20 * 18 * * 70 16 14 60 12 50 10 40 8 IB: TRIM45 30 TRIM45 6 20 IB: β-actin 4 2 10 0 0 Duration of 015 0015 15 0 15 015 0015 15 0 15 serum induction (min)

EGR1 300 12 * 250 sh-TRIM45#3 * 10 * * sh-TRIM45#2 200 * 8 150 * sh-TRIM45#1 100 IB: EGR1 6 sh-control 50 IB: TRIM45 4 0 Duration of 015 0015 15 0 15 IB: β-actin serum induction 2 (min)

0 0 24 48 72 Time (h)

c-Jun EGR1 30 160 * * 140 25 Δ HA-PKC: NPS WT 120 20 Flag-TRIM45: 100

IB: EGR1 15 80 60 10 IB: HA 40 5 IB: Flag 20 Duration of 0 0 IB: β-actin serum induction 015 015 015 015 (min) Calphostin C (500nM)

© 2015 Macmillan Publishers Limited Oncogene (2015) 1280 – 1291 TRIM45 regulates activation of RACK1/PKC pathway T Sato et al 1288 important role in the crosstalk between PKC–RACK1, ERK and JNK On the basis of our results showing that expression of TRIM45 is pathways under unique conditions. Thus, to obtain a clue for the induced after serum stimulation and that it participates in possibility that TRIM45 modulates expression levels of proteins inhibiting cell proliferation via the RACK1–PKC-mediated signaling that are involved in these pathways, we performed immunoblot pathway, we hypothesize that the components of AP-1 transcrip- analysis in HeLa cells transfected with si-TRIM45 #3 used in the tion factor bind to the promoter region of the TRIM45 gene, above-described experiments. We checked the expression levels resulting in increase in the expression of TRIM45 in order to of c-Jun, phospho-JNK, JNK1, JNK2, phospho-MEK1/2, phospho- negatively regulate the RACK1–PKC signaling pathway. Our ChIP Erk1/2 and Erk1/2 at the indicated times after serum induction. study revealed that Fos binding to the promoter region of the Immunoblot analysis showed that expression levels of c-Jun, TRIM45 gene is significantly enhanced by serum stimulation. This phospho-JNK, JNK1, JNK2, phospho-MEK1/2 and phospho-Erk1/2 finding is consistent with our results showing that serum or PMA were promptly elevated after serum stimulation with silencing of stimulation significantly enhances the protein expression of TRIM45 compared with the levels in control cells (Figure 6b). TRIM45. Moreover, we confirmed that the expression levels of phospho- Recently, the signaling pathway regulating AP-1 activity has JNK and JNK1 were changed by exogenously expressed TRIM45. been reported to be upregulated in many carcinoma cells. A Following serum stimulation, the expression of phospho-JNK and number of studies have shown the importance of highly active JNK1 was slightly suppressed in HeLa cells in which Flag-TRIM45 AP-1 signaling in carcinogenesis.41 Both the ERK and JNK was stably expressed compared with the levels in control cells pathways are thought to contribute to progression of the cell (Figure 6c). The observations that transcription of TRIM45 division cycle via cyclin-dependent kinases in cancer cells and was significantly elevated after serum stimulation led us to simultaneous activation of these pathways is essential in speculate that TRIM45 is one of the members that participate in tumorigenesis. However, there is precedent for the idea that the construction of the negative feedback loop of the PKC-mediated JNK pathway is mainly activated by stress stimulation. On the basis rewired ERK–JNK signaling pathway. We performed chromatin of the results of recent studies, especially in human melanomas in immunoprecipitation (ChIP) assay at the promoter region of the which activity of the ERK pathway is upregulated, a model of TRIM45 gene in HeLa cells in order to determine whether c-Fos crosstalk between the JNK–Jun pathway and the MEK–ERK binds to putative promoter regions of the TRIM45 gene and signaling pathway has been proposed.12 It has also been activates its transcription. In this assay, we detected that c-Fos recognized that PKC has versatile roles in provoking a strong bound to the promoter region of the TRIM45 gene. In addition, JNK signal by ERK. Increasing amounts of c-Jun, caused by interaction between c-Fos and the promoter region of the phosphorylation of ERK, leads to activation of the AP-1 complex TRIM45 gene was significantly enhanced after serum stimulation and causes increased expression of RACK1. RACK1 binds to (Figure 6d). activated PKC and together they associate with JNK, which, in turn, causes the phosphorylation of JNK. The strengthened activity of phosphorylated JNK together with increased expression of c-Jun DISCUSSION leads to phosphorylation of c-Jun, which constitutively accelerates In this study, results of several assays showed that TRIM45 is a expression of yet more RACK1. In this way, a feed-forward novel RACK1-interacting protein, and a more profound molecular amplification loop of the rewired ERK–JNK signaling pathway is function of TRIM45 in cell proliferation was also revealed. RACK1 is established (Figure 7).12,42,43 This deliberate crosstalk mechanism known as an adaptor protein that has versatile roles in organizing is present not only in melanomas but also in many other human kinases involved in PKC and MAPK signaling pathways around its cancers. Increased Fos-Jun AP-1-binding activity, resulting in substrates in order to optimize accurate phosphorylation of the deregulation of Jun family members, has also been observed in substrates and regulate their activity. Although RACK1 is not human cervical cancer.41,44 Although the detailed mechanism that substrate for specific enzymes including PKC, substrate phosphor- mediates between the MEK–ERK cascade and Stress-activated ylation in which PKC participates is enhanced in the presence of protein kinase cascade remains unknown, our study revealed the RACK1. Given our results showing that TRIM45 downregulates mechanisms underlying downregulation of this rewired ERK–JNK transcription of genes downstream of RACK1 in response to serum signaling pathway by TRIM45. Together with our observation that stimulation, it is possible that TRIM45 acts as an E3 ligase for the binding of Fos protein to the promoter region of the TRIM45 gene degradation of RACK1. However, our findings did not strongly was significantly enhanced after serum stimulation, these findings suggest that RACK1 is a target of TRIM45-mediated proteosomal raise the possibility that transcription of the TRIM45 gene is degradation. induced to give rise to a negative feedback function against RACK1 was originally identified as an anchoring protein for excess progression of cell proliferation and tumorigenesis. On the activated PKC; therefore, we propose that TRIM45 directly interacts other hand, our findings from the experiment using a PKC with RACK1 and inhibits RACK1–PKC interaction for inactivation of inhibitor (Figure 5h) may not be able to completely explain that RACK1. In this study, we revealed that TRIM45 attenuates the TRIM45 regulates the expression of c-Jun through specifically interaction between RACK1 and PKCβII in a TRIM45 dose- inhibiting the PKC–RACK1 signaling pathway. TRIM45 directly dependent manner. We also found that inhibition of the interacts with RACK1, but not with PKC, suggesting that TRIM45 is interaction between RACK1 and PKCβII by TRIM45 occurs relatively required for other RACK1 signal transduction regulating expres- early after serum stimulation, supporting our speculation that sion of AP-1 genes even if the activity of PKC is inhibited. In turn, inactivation of RACK1 depends on direct interference by TRIM45 as calphostin C decreased the expression of c-Jun and EGR1 only rather than post-translational modifications such as ubiquitylation. in cells in which TRIM45 was depleted, it is also possible that PKC So far, little is known about the molecules that participate in activity was strongly inhibited in the presence of TRIM45. negative regulation of RACK1–PKC interaction. In addition to the Although our hypothesis remains a matter of speculation, our fact that TRIM45 directly interacts with RACK1, it is interesting that finding raises the possibility that TRIM45 influences signal the critical domain for interacting between RACK1 and PKC is also transduction in which RACK1 is involved. There is still a veiled required for interaction with TRIM45. It has been shown that mechanism in this pathway, and we will try to clarify the reason introduction of peptides, generated from the specific domains for these results in the future. of PKC, into cells can inhibit various PKC-mediated cellular The results of this study also indicated the possibility that functions.40 The results of this study raise the possibility that a TRIM45 has crucial functions other than its functions in cell growth certain small molecule including TRIM45 can also specifically and carcinogenesis. We found that TRIM45 interacts with RACK1 in inhibit RACK1–PKC interaction with potential therapeutic benefit. certain stress situations. In summary, our study revealed a

a TRIM45 TRIM45 b si-TRIM45: – + 12 120 p<0.05 serum induction (min): 0 30 60 0 30 60 10 Mr (k) 100 43 c-Jun 8 80 IB: c-Jun P-JNK 6 60 43 IB: P-JNK 4 57 40 JNK2 Relative mRNA levels 2 Relative mRNA levels 20 IB: JNK2 JNK1 0 0 43 PMA(5nM) –+serum –+ IB: JNK1 induction P-MEK1/2 43 IB:TRIM45 IB:TRIM45 IB: P-MEK1/2 43 P-Erk1/2 IB:β-actin IB:β-actin IB: P-Erk1/2 43 Erk1/2 IB: Erk1/2 TRIM45 IB: TRIM45 β-actin IB: β-actin

c TRIM45 Flag-TRIM45: – + d p<0.05 0.10 seruminduction(min): 0 60 0 60 0.09 P-JNK 0.08 0.07 IB: P-JNK 0.06 JNK1 0.05

ChIP / Input 0.04 IB: JNK1 0.03 Flag-TRIM45 0.02 0.01 IB: Flag 0 β-actin IgG c-Fos IgG c-Fos IB: β-actin serum –+ induction TRIM45

-1160 +1 +589 Exon1 ATG Pro Primer Figure 6. Effect of knockdown of TRIM45 on the expression of ERK/JNK/c-Jun and recruitment of c-Fos to the promoter of the TRIM45 gene after serum stimulation. (a) PMA and serum stimulation enhanced transcription of the TRIM45 gene in HeLa cells. Data are means of values from three independent experiments. Lower panels show results of immunoblot analysis of TRIM45 with or without stimulation by PMA and serum. Anti-β-actin antibody was used as an internal control. (b) TRIM45 siRNA affects protein levels of MEK/ERK and JNK pathway in HeLa cells. Cell lysates introduced with non-targeting siRNA or with siRNA specific for human TRIM45 (si-TRIM45) were analyzed by western blots using the indicated antibodies. (c) Exogenously expressed TRIM45 suppresses protein expression of phospho-JNK under the condition of serum stimulation. Results of immunoblot analysis of endogenous phospho-JNK and JNK1 in HeLa cells in which Flag-TRIM45 or mock was stably expressed are shown. (d) Occupancy of c-Fos on the promoter of the TRIM45 gene with or without serum stimulation in HeLa cells. IgG indicates control IgG. ChIP/Input is the average from triplicate; error bars show s.d. PCR product size is 102 bp. Pro in the schema indicates the promoter region. negative feedback mechanism for the rewired ERK–JNK signaling assays, western blotting and ChIP, we washed cell cultures at 50–70% pathway in cancer cells by TRIM45 through RACK1-mediated confluency twice with phosphate-buffered saline, starved them in serum- interruption. Further studies aimed at verification of the profound free medium for >12 h and then treated them with serum-containing functions of TRIM45 in MAPK signaling pathways should be medium for the times indicated in each figure. helpful for advances in cancer biology and therapy. Antibodies and reagents MATERIALS AND METHODS For western blot analysis, the following antibodies were used: anti-FLAG (1 μg/ml; M2 or M5; Sigma); anti-HA (1 μg/ml; HA.11; Covance, Princeton, Cell culture and treatment NJ, USA); anti-HA (1 μg/ml; Y11; Santa Cruz Biotechnology, Santa Cruz, CA, HEK293T, HeLa and COS-7 cells (ATCC, Manassas, VA, USA) were cultured USA); anti-TRIM45 (1 mg/ml; ab84303; Abcam, Cambridge, UK); anti-RACK1 under an atmosphere of 5% CO2 at 37 °C in Dulbecco’s modified Eagle’s (1:1000, Cell Signaling Technology (CST), Danvers, MA, USA); anti-GST (0.2 medium (Sigma, St Louis, MO, USA) supplemented with 10% (v/v) fetal mg/ml; B-14; Santa Cruz Biotechnology); anti-phosphorylated PKC (pan) bovine serum (Invitrogen, Carlsbad, CA, USA), 55 μM β-mercaptoethanol (1:1000, CST); anti-c-Jun (1:1000, CST); anti-EGR1 (1:1000, Santa Cruz (Gibco Invitrogen Corp., Paisley, UK), 2 mML-glutamine, 0.1 mM minimum Biotechnology); anti-phosphorylated Stress-activated protein kinase/JNK essential medium non-essential amino acid, penicillin (10 U/ml) and (1:1000, CST); anti-JNK1 (1:1000, CST); anti-JNK2 (1:1000, CST); anti- streptomycin (0.1 mg/ml). Parental HeLa S3 cells (Invitrogen) and its phosphorylated MEK1/2 (1:1000, CST); anti-PKC (0.2 mg/ml; A-3; Santa derivative was cultured as described previously.45 Stable overexpression of Cruz Biotechnology); anti-phosphorylated Erk1/2 (1:1000, CST); anti- Flag-TRIM45 proteins was obtained by retroviral infection using pMX-puro phospho-PKC substrate antibody (1:1000, CST); anti-Erk1/2 (1:1000, CST); expression plasmid followed by puromycin (Sigma) selection as anti-His6 (0.2 μg/ml; H-15; Santa Cruz Biotechnology); anti-β-actin (0.2 μg/ described.46 For serum induction assay, as stimulation of growth- ml; AC15; Sigma) and anti-Myc (1 μg/ml; 9E10; Covance). Final concentra- promoting extracellular stimuli in quantitative reverse transcriptase–PCR tions of the reagents were as follows: 10 or 30 μM Pseudo RACK1 (TOCRIS

TPA, growth factors (VECTOR), and then photographed with a CCD camera (DP71, Olympus, Tokyo, Japan) attached to an Olympus BX51 microscope.

RNA interference We generated stable clones constitutively expressing a shRNA targeting PKC PKC P inactive PKC TRIM45 (sh-TRIM45) using pSUPER-retro-puro vector (OligoEngine, Seattle, ERK WA, USA). pSUPER-retro-puro containing an sh-TRIM45 sequence PKC (sh-TRIM45 #1, 5′-GCTGGGCTTTGTAAGCAAACT-3′; sh-TRIM45 #2, 5′-GGTGG TRIM45 RACK1 TRIM45 AGTGAAGGCTTTAACC-3′; sh-TRIM45 #3, GCTCAGGAAGCTGAACAAAGT-3′) RACK1 was constructed according to the manufacturer’s protocol. Approximately P RACK1 50% conﬂuent HEK293T cells in 100 mm dishes were co-transfected with JNK μ TRIM45 10 g pSUPER-retro-puro-TRIM45 or scrambled shRNA vector along with × 10 μg amphotrophic packaging plasmid pCL10A1 using Fugene HD c-Fos c-Jun reagent (Roche). At 48 h after transfection, the culture supernatant containing the retrovirus was collected, and the retroviral supernatant P P was added to HeLa cells in 60-mm dishes with polybrene (8 μg/ml, Sigma). Cells were cultured with puromycin (1 μg/ml). After selection in a medium cell proliferation, containing puromycin, the resulting cell lines were checked by immuno- P P tumorigenesis, .... TRIM45 blot analysis with anti-TRIM45 antibody. For transient TRIM45 knockdown in HeLa cells, we used siRNAs targeting human TRIM45 (Invitrogen, Stealth Figure 7. Model for effect of TRIM45 on rewired ERK–JNK pathways. RNAi, Life Technologies, Paisley, UK; 10620318, 10620319) or siGENOME Upon extracellular stimuli, the PKC and RACK1 complex phospho- NON-TARGETING siRNA Pool (Dharmacon and Thermo Fisher Scientiﬁc, rylates JNK and provokes strong JNK signaling, resulting in Waltham, MA, USA; D-001206-14) using Lipofectamine RNAiMAX Transfec- phosphorylation of c-Jun. Phosphorylated c-Jun promotes transcription Reagent (Invitrogen) according to the manufacturer’s protocol and as tion of the RACK1 gene, which causes enhancement of a feed- described previously.45 forward loop. On the other hand, increasing amounts of c-Jun, caused by phosphorylation of ERK, leads to activation of the AP-1 complex and causes c-Jun stability, resulting in enhanced expression Real-time quantitative PCR of RACK1. Conversely, increasing amounts of c-Jun and c-Fos cause Total RNA was isolated from HeLa cells using ISOGEN II (Nippon Gene, recruitment of c-Fos to the promoter of the TRIM45 gene and Toyama, Japan), followed by reverse transcription by ReverTra Ace enhance the expression of TRIM45, and TRIM45 in turn interacts (Toyobo, Osaka, Japan). The resulting complementary DNA was subjected with RACK1 to inhibit RACK1–PKC interaction. to real-time PCR with a StepOne machine and Power SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA). The average Bioscience, Bristol, UK), 5 nM for PMA (Sigma), 0.5 mM for arsenite threshold cycle (Ct) was determined from independent experiments and (71287; Fluka, Ronkonkoma, NY, USA), human MBP (Sigma), and 500 nM the level of gene expression relative to human ACTB was determined. The for calphostin C (Sigma). primer sequences are shown in Supplementary Materials and methods.

Recombinant protein and in vitro binding assay ChIP assay GST-fused proteins including deletion mutants of human RACK1 were We performed ChIP as previously described.45 Immunoprecipitated and expressed in XL-10 cells using pGEX plasmid vector (GE Healthcare, input material was analyzed by quantitative PCR. ChIP signal was Piscataway, NJ, USA) and then purified by reduced glutathione-sepharose normalized to total input. The primer sequences of the promoter region beads (GE Healthcare). His6-Flag-tagged TRIM45 was expressed in the Sf9 of the TRIM45 gene used for ChIP-quantitative PCR are 5′-AGGAA insect cell line using a baculovirus protein expression system (Invitrogen). TCAAGTGAAACAGTCGCCC-3′ and 5′-TCCAGTTGCGTCAGCTTGTCTACT-3′. His6-HA-tagged PKCβII was expressed in Escherichia coli strain BL21 (DE3) (Invitrogen) and then purified by using ProBond metal affinity beads (Invitrogen). In vitro binding assays were performed as described In vitro kinase assay 47 previously. HeLa cell lines stably expressing Flag-tagged TRIM45 or mock cells were used, and cellular proteins were extracted by cell lysis in PKC extraction Cell proliferation assay buffer (40 mM HEPES-KOH, pH 7.9, 150 mM NaCl, 0.5% Triton X-100 and 1.5 mM MgCl2) that contained protease inhibitors and phosphatase One hundred thousand HeLa cells stably expressing sh-TRIM45 clones or inhibitors. PKC was immunoprecipitated with antibodies for 2 h at 4 °C. scrambled shRNA as a negative control were seeded on 60 mm dishes and Protein A-sepharose (GE Healthcare) that had been equilibrated with the ’ fi ’ cultured in Dulbecco s modi ed Eagle s medium containing 10% (v/v) fetal same solution was added to the mixture, which was then tumbled for 1 h bovine serum. The number of live cells was determined by counting using at 4 °C. The resin was separated by centrifugation and washed five times a hemocytometer after Trypan blue staining at the indicated times. Data with ice-cold lysis buffer and then twice with kinase buffer (40 mM HEPES shown are average values from three independent experiments. (pH 7.9), 10 mM MgCl2,10mM CaCl2 and 3 mM MnCl2). The kinase assay was initiated by adding kinase buffer containing 10 μM radiolabeled 32 μ Immunofluorescence staining and microscopic observation of [gamma- P] adenosine triphosphate, human MBP (0.5 g) and PMA (10 stress granules ng/ml). Reactions were performed at 30 °C for 30 min. The reactions were terminated by adding sodium dodecyl sulfate sample buffer and boiled for COS-7 cells grown on glass coverslips were transfected with plasmids using 5 min. The reaction products were analyzed by sodium dodecyl sulfate– Fugene HD reagent (Roche, Branchburg, NJ, USA). Thirty-six hours after polyacrylamide gel electrophoresis, autoradiography and western blotting. transfection, the cells were stimulated for stress granule assembly with 0.5 mM sodium arsenite for 60 min. Cells expressing HA-RACK1 with and without Flag-tagged TRIM45 or cells expressing Flag-TRIM45 without HA- Statistical analysis fi RACK1 grown on glass coverslips were xed for 10 min at room The statistical significance of the difference between mean values was temperature with 2% formaldehyde in phosphate-buffered saline and tested using Student’s t-test. Data are means ± s.d. then incubated for 1 h at room temperature with a primary antibody to Flag, HA or RACK1 in phosphate-buffered saline containing 0.1% bovine serum albumin and 0.1% saponin. They were then incubated with Alexa488-labeled goat polyclonal antibody to mouse IgG or Alexa546- labeled goat polyclonal antibody to rabbit IgG (Molecular Probes, Eugene, CONFLICT OF INTEREST OR, USA) at a dilution of 1:1000, covered with a drop of VECTASHIELD The authors declare no conflict of interest.

Oncogene (2015) 1280 – 1291 © 2015 Macmillan Publishers Limited TRIM45 regulates activation of RACK1/PKC pathway T Sato et al 1291 ACKNOWLEDGEMENTS 22 Bauman AL, Scott JD. Kinase- and phosphatase-anchoring proteins: harnessing We thank Dr Toshio Kitamura (Tokyo University) for the plasmid and cell lines. This work the dynamic duo. Nat Cell Biol 2002; 4: E203–E206. was supported in part by a research grant from Grant-in-Aid for Scientific Research from 23 Lopez-Bergami P, Habelhah H, Bhoumik A, Zhang W, Wang LH, Ronai Z. RACK1 19 the Ministry of Education, Culture, Sports, Science and Technology in Japan, Japan mediates activation of JNK by protein kinase C [corrected]. Mol Cell 2005; : Foundation for Applied Enzymology, and Children’s Cancer Association of Japan. 309–320. 24 Lopez-Bergami P, Ronai Z. Requirements for PKC-augmented JNK activation by MKK4/7. Int J Biochem Cell Biol 2008; 40:1055–1064. AUTHOR CONTRIBUTIONS 25 Nisole S, Stoye JP, Saib A. TRIM family proteins: retroviral restriction and antiviral defence. Nat Rev Microbiol 2005; 3:799–808. TS conceived and designed experiments, performed all analysis, analyzed 26 Reymond A, Meroni G, Fantozzi A, Merla G, Cairo S, Luzi L et al. The tripartite motif results and wrote the paper; HT conceived and designed experiments, family identifies cell compartments. EMBO J 2001; 20:2140–2151. performed a part of the experiments and analyzed results; SH analyzed results; 27 Meroni G, Diez-Roux G. TRIM/RBCC, a novel class of 'single protein RING finger' E3 27 – AI analyzed results; and TA conceived and designed experiments and analyzed ubiquitin ligases. Bioessays 2005; : 1147 1157. results. 28 Noguchi K, Okumura F, Takahashi N, Kataoka A, Kamiyama T, Todo S et al. TRIM40 promotes neddylation of IKKgamma and is downregulated in gastrointestinal cancers. Carcinogenesis 2011; 32: 995–1004. 29 Sato T, Okumura F, Kano S, Kondo T, Ariga T, Hatakeyama S. 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