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T cell development involves TRAF3IP3-mediated ERK signaling in the Golgi

Zou et al. identify a Golgi-associated factor, TRAF3-interacting protein 3 (TRAF3IP3), as a crucial mediator of thymocyte development regulating TCR-stimulated ERK signaling in the Golgi.

The development of T cells in the thymus involves sequential progression of thymocytes through different stages, which can be defined based on their surface expression of CD4 and CD8 coreceptors (Germain, 2002). During the early CD4  CD8  double-negative (DN) stages, DN1-DN4, the gene encoding the TCR chain undergoes rearrangement, leading to the formation of a pre-TCR that drives the further differentiation of the cells into CD4 + CD8 + double-positive (DP) stage (Michie and Zúñiga-Pflücker, 2002). Subsequently, rearrangement of the TCR gene occurs in the CD4 + CD8 + DP stage, leading to formation of surface  TCR. DP thymocytes are then subject to positive and negative selections by complexes of self-peptide and MHC displayed on thymic epithelial cells and other antigen-presenting cells (Klein et al., 2014). The majority of DP thymocytes die of "neglect" caused by expression of TCRs that fail to recognize self-peptide-MHC complexes, and those that bind self-peptide-MHC complexes with high affinity die of negative selection. Only the thymocytes with TCRs that bind self-peptide-MHC complexes with intermediate strength are positively selected to become mature CD4 + or CD8 + single-positive (SP) thymocytes (Germain, 2002;Klein et al., 2014).
Signal transduction from the TCR plays a crucial role in the progression of thymocytes from the DP to SP stages (Starr et al., 2003;Zamoyska and Lovatt, 2004;Wang et al., 2010). In particular, the MAPK signaling cascade is critically required for the development and maturation of thymocytes (Pagès et al., 1999;Alberola-Ila and Hernández-Hoyos, 2003;Fischer et al., 2005;Kortum et al., 2013). This signaling cascade involves sequential activation of the small G protein Ras and its downstream kinases RAF, mitogen/extracellular signal-regulated kinase (MEK), and extracellular signal-regulated kinase (ERK; Kortum et al., 2013). Among the RAF members, BRAF is particularly important for TCR-stimulated MEK-ERK activation and thymocyte development (Tsukamoto et al., 2008;Dillon et al., 2013). TCR stimulation leads to the activation of BRAF, but not RAF1, establish TRAF3IP3 as a novel regulator of thymocyte development and suggest a compartmentalized BRAF-MEK-ERK signaling mechanism regulated by TRAF3IP3.

TRAF3IP3 is required for thymocyte development
To assess the function of TRAF3IP3 in the immune system, we analyzed its expression at the protein level and found that TRAF3IP3 was highly expressed in the thymocytes, most abundantly in the CD4 + CD8 + DP thymocytes ( Fig. 1 A). This finding prompted us to examine the role of TRAF3IP3 in thymocyte development. We generated germline Traf3ip3 KO mice by crossing the Traf3ip3-targeted mice with CMV-Cre mice (Fig. 1, B-D; Schwenk et al., 1995). We also crossed the Traf3ip3-targetd mice with FRT deleter (Rosa26-FLPe) mice to generate Traf3ip3-flox mice, which were further crossed with Cd4-Cre mice to produce Traf3ip3 T cell-conditional KO (TKO) mice ( Fig. 1, B, E, and F). The Traf3ip3-KO mice were healthy and fertile (not depicted), and these mutant mice and their WT control mice had similar numbers of total thymocytes ( Fig. 2 A). However, the Traf3ip3-KO mice had considerably lower frequencies of CD4 + and CD8 + SP thymocytes, and concomitantly higher frequencies of DP thymocytes, in thymocytes (Dillon et al., 1991). Consistently, BRAF is required for TCR-stimulated ERK activation and DP thymocyte positive selection; however, how BRAF mediates activation of MEK and ERK in thymocytes is incompletely understood (Tsukamoto et al., 2008;Dillon et al., 2013).
In the present study, we identified TRAF3-interacting protein 3 (TRAF3IP3) as a novel regulator of MAPK signaling and thymocyte development. TRAF3IP3, also called TRAF3-interacting JNK-activating modulator (T3JAM), was originally identified as a protein that interacts with TRAF3 and synergizes with TRAF3 to activate JNK under overexpression conditions (Dadgostar et al., 2003). TRAF3IP3 mRNA is specifically expressed in lymphoid organs, although its physiological role has not been investigated (Dadgostar et al., 2003). We found that TRAF3IP3 is highly expressed in DP thymocytes and associated with the Golgi apparatus. Using newly generated Traf3ip3 KO mice, we demonstrated a crucial role for TRAF3IP3 in mediating the thymocyte development from DP to SP stages. The TRAF3IP3 deficiency specifically attenuates TCR-stimulated activation of MEK and its downstream kinase ERK. TRAF3IP3 mediates MEK activation by recruiting MEK to the Golgi and, thereby, facilitating MEK interaction with its upstream kinase BRAF. These findings (B) Schematic picture of Traf3ip3 gene targeting strategy using an FRT-LoxP vector, with genotyping primers indicated (P1-P5). Chimeric mice were crossed with a CMV-Cre mouse to generate germline Traf3ip3-KO mice or with an FRT deleter (Rosa26-FLPe) mouse to generate Traf3ip3-flox mice, which were subsequently crossed with a Cd4-Cre mouse to produce Traf3ip3-TKO mice. (C) Genotyping PCR using P1/P2 primer pair for the WT allele and P5/P4 primer pair for the KO allele. (D) Immunoblot analysis of TRAF3IP3 and Actin using thymocytes of WT and Traf3ip3-KO (KO) mice. (E) Genotyping PCR to amplify the Traf3ip3-flox (using P3/P4 primer pair) and WT (using P1/P2 primer pair) alleles (top) or the Cre cDNA (bottom). (F) Immunoblot analysis of TRAF3IP3, showing T cell-specific TRAF3IP3 ablation. PCR primer sequences are listed in Table S1. Data are representative of four independent experiments with at least six mice of each genotype (A, D, and F).
mice had fewer naive T cells than WT mice, whereas the number of memory T cells in both genotypes was normal (Fig. 2 E). In contrast, the TRAF3IP3 deficiency did not affect the than the WT mice (Fig. 2, B and C). The Traf3ip3-KO mice also had a reduced frequency and number of splenic CD4 + and CD8 + T cells (Fig. 2, D and E). In addition, the Traf3ip3-KO  thymocyte development at the DN stages, DN1-DN4 (Fig. 2, F and G). These data suggest that TRAF3IP3 is required for thymocyte development during the DP to SP transition. We also examined the development of regulatory T (T reg) cells,  T cells, and invariant NKT (iNKT) cells in the Traf3ip3-KO mice. The Traf3ip3-KO mice had a reduced number of thymic and splenic T reg cells and an increased number of splenic  T cells, whereas the number of iNKT cells was comparable between the KO and WT mice (Fig. 2 H). The defect of the Traf3ip3-KO mice in T reg cell development was consistent with their impaired production of SP thymocytes, and how TRAF3IP3 regulated the peripheral  T cells was unclear.

TRAF3IP3 has a cell-intrinsic role in regulating thymocyte development
To examine whether the thymocyte development defect of Traf3ip3-KO mice is cell intrinsic, we generated mixed BM chimeric mice by transferring BM cells derived from WT or Traf3ip3-KO mice (CD45.2 + ), along with BM cells derived from WT B6.SJL mice (CD45.1 + ), into the lymphocytedeficient Rag1-KO mice. The chimeric mice had a similar percentage of thymocytes derived from the CD45.1 + and CD45.2 + BM cells (Fig. 3 A). Furthermore, the CD45.1 + and CD45.2 + WT thymocytes displayed a similar profile of development ( Fig. 3 A). However, under the same conditions, the CD45.2 + Traf3ip3-KO BM cells yielded a substantially Barnden et al., 1998). The OT-I and OT-II TCRs positively select OVA-specific CD8 + and CD4 + thymocytes, respectively. We crossed Traf3ip3-KO mice with OT-I or OT-II mice to generate Traf3ip3-KO OT-I or Traf3ip3-KO OT-II mice. The Traf3ip3-KO OT-I and Traf3ip3-KO OT-II mice had a substantially reduced frequency and number of CD8 + and CD4 + SP thymocytes, respectively, than their corresponding control (Traf3ip3 +/+ OT-I and Traf3ip3 +/+ OT-II) mice (Fig. 5, A and B). Consistently, the Traf3ip3-KO OT-I and Traf3ip3-KO OT-II mice also had a reduced frequency of CD8 + and CD4 + splenic T cells, respectively (Fig. 5 C). Flow cytometry analysis for the transgenic TCR chain V2 indicated that nearly all CD8 + SP thymocytes and splenic CD8 + T cells expressed the OVA-specific TCR in WT and Traf3ip3-KO OT-I mice, although lower V2 levels could be detected in the TRAF3IP3-deficient CD8 + SP thymocytes and splenic CD8 + T cells (Fig. 5 D). Similar results were obtained in the analysis of Va2 expression in CD4 + SP thymocytes and splenic CD4 + T cells of the WT and Traf3ip3-KO OT-II mice (Fig. 5 E).
To examine the role of TRAF3IP3 in thymocyte negative selection, we crossed the Traf3ip3-KO mice with H-Y mice expressing a transgenic TCR specific for the MHCI-restricted male antigen H-Y (Kisielow et al., 1988). In male H-Y mice, CD4 + CD8 + DP thymocytes undergo negative selection induced by endogenous H-Y antigen (Kisielow et al., 1988). The male WT and Traf3ip3-KO H-Y TCR transgenic mice displayed similar frequencies and absolute numbers of total, DP, and CD8 + SP thymocyte populations (Fig. 5, F and G). Analysis of the female mice, in which thymocytes were positively selected, revealed a substantial reduction in the CD8 + SP thymocytes (Fig. 5, F and G), thus further confirming the requirement of TRAF3IP3 for positive selection. Together, these data demonstrate a critical role for TRAF3IP3 in regulating thymocyte development at the stage of positive selection.

TRAF3IP3 mediates TCR-stimulated MEK-ERK activation
Because thymocyte positive selection is critically dependent on TCR signaling (Alberola-Ila and Hernández-Hoyos, 2003), we examined the role of TRAF3IP3 in regulating DP thymocyte signaling after TCR cross-linking with anti-CD3 plus anti-CD4. The Traf3ip3 +/+ and Traf3ip3-KO DP thymocytes had comparable phosphorylation of several TCRproximal factors, Lck, ZAP70, and PLC-, as well as the downstream MAPKs p38 and JNK and the IB kinase (IKK) substrate IB (Fig. 6, A and B). The TRAF3IP3 deficiency also did not influence the activation of the calcium-dependent transcription factor NFAT (Fig. 6 C). However, the Traf3ip3-KO DP thymocytes had a severe defect in the induction of ERK phosphorylation (Fig. 6 B). This result was not caused by reduced frequency of CD69 hi thymocytes in the Traf3ip3-KO mice because impaired ERK phosphorylation was also detected in purified CD69-negative Traf3ip3-KO DP thymocytes (Fig. 6 D). The TRAF3IP3 deficiency also attenuated the inducible phosphorylation of the ERK kinase MEK (Fig. 6 E). Thus, TRAF3IP3 has an important lower frequency of CD4 + and CD8 + SP thymocytes and of splenic T cells than the WT BM cells, although the KO and WT BM cells produced a similar frequency of DN cells (Fig. 3, A-D). This result suggested a cell-intrinsic role for TRAF3IP3 in regulating thymocyte development.
To further confirm the cell-intrinsic function of TRAF3IP3 in thymocyte development, we used the Traf3ip3-TKO mice generated by crossing the Traf3ip3-flox mice with Cd4-Cre mice, which is known to delete loxP-flanked genes from the very late DN stage of thymocyte development (Fig. 1 B ;Wolfer et al., 2002). The Traf3ip3-TKO and WT control mice had comparable numbers of total thymocytes (Fig. 3 E). However, as seen with the Traf3ip3-KO mice, the Traf3ip3-TKO mice had a significantly reduced frequency of CD4 + and CD8 + SP thymocytes and a concomitantly increased frequency of DP thymocytes (Fig. 3, F and G). Traf3ip3-TKO mice also had fewer splenic CD4 + and CD8 + T cells than WT mice (Fig. 3, H and I). These data further emphasize a cell-intrinsic defect of TRAF3IP3-deficient thymocytes in the transition from DP to SP stages.

TRAF3IP3 regulates thymocyte positive selection
The development of thymocytes from DP to SP stages involves a process of positive selection, which is guided by TCR stimulation by self-peptide-MHC complexes (Alberola-Ila and Hernández-Hoyos, 2003). A hallmark of positive selection is up-regulation of surface TCR and CD69 (Swat et al., 1993;Yamashita et al., 1993;Vanhecke et al., 1997;Nakayama et al., 2002). To more precisely define the stage of developmental block in the Traf3ip3-KO thymocytes, we analyzed the thymocyte populations based on the expression of CD69 and TCR. This method could separate the thymocytes into four populations, representing preselection DP thymocytes (population 1; TCR lo CD69 lo ), a transitional population initiating positive selection (population 2; TCR int CD69 hi ), a population immediately after positive selection (population 3; TCR hi CD69 hi ), and a more mature postselection SP thymocyte population (population 4; TCR hi CD69 lo ; Hu et al., 2012). Compared with their Traf3ip3 +/+ littermates, the Traf3ip3-KO mice had an increased frequency of preselection DP thymocytes (population 1) and concomitant reduction in the subsequent populations, most dramatically the postselection population 3 and population 4 cells (Fig. 4, A and B). Accordingly, the Traf3ip3-KO mice had significantly fewer mature SP thymocytes in populations 3 and 4 than WT mice (Fig. 4 C). Further analysis revealed a substantially reduced frequency and absolute number of CD69 hi cells within the total DP thymocyte population and the CD4 + CD8 int transitional thymocyte population of Traf3ip3-KO mice (Fig. 4, D and E). The similar results were obtained from thymocyte analysis of the Traf3ip3-TKO mice (not depicted). These results suggested a defect of the TRAF3IP3-deficient thymocytes in positive selection.
To further examine the role of TRAF3IP3 in regulating thymocyte positive selection, we used TCR transgenic mice expressing an OVA-specific TCR, restricted by MHC class I (OT-I) or MHC class II (OT-II; Hogquist et al., 1994; mutant thymocytes with a constitutively active form of MEK (MEK1DD) by crossing the Traf3ip3-TKO mice with R26Stop FL MEK1DD mice expressing MEK1DD under the control of Cre (Srinivasan et al., 2009). MEK1DD transgene was expressed in CD4 + and CD8 + T cells in the progeny mice ( Fig. 7 A and not depicted), and the deletion of TRAF3IP3 was confirmed in the CD4 + SP thymocytes (Fig. 7 B). The Cd4-Cre-directed expression of MEK1DD in WT mice did not significantly alter the frequency or number of SP thymocytes (Fig. 7, C and D). However, expression of this constitutively active MEK in Traf3ip3-TKO mice largely restored role in mediating TCR-stimulated activation of the MEK-ERK signaling pathway.

Constitutively active MEK rescues the thymocyte development defect
The signaling cascade leading to activation of MEK and ERK is required for thymocyte development (Pagès et al., 1999;Alberola-Ila and Hernández-Hoyos, 2003;Fischer et al., 2005;Kortum et al., 2013). To directly examine the functional significance of attenuated MEK-ERK phosphorylation in TRAF3IP3-deficient thymocytes, we rescued these  These results suggest that TRAF3IP3 mediates TCR-stimulated MEK-ERK activation by facilitating the inducible interaction of BRAF with MEK.
We surmised that TRAF3IP3 might function as a scaffold protein facilitating the BRAF-MEK interaction. Coimmunoprecipitation assays revealed constitutive binding of TRAF3IP3 with MEK in both DP thymocytes and transfected HEK293 cells (Fig. 8, C and D), and this molecular interaction was also indicated by the colocalization of TRAF3IP3 and MEK in confocal microscopy (Fig. 8 E). However, we were unable to detect appreciable binding between TRAF3IP3 and BRAF (not depicted), suggesting that TRAF3IP3 might not function as a simple adaptor. Domain analysis of TRAF3IP3 using the SMART program (Schultz et al., 1998) revealed a transmembrane domain located at its C terminus (Fig. 8 F). Subcellular fractionation assays confirmed the predominant localization of TRAF3IP3 in the membrane fraction (Fig. 8 G). MEK1 was also partially localized to the membrane fraction, and the level of membrane-associated MEK1 was reduced in the the population of SP thymocytes (Fig. 7, C and D), as well as splenic T cells (Fig. 7, E and F). These findings emphasized a role for TRAF3IP3-mediated ERK signaling in mediating thymocyte development.

TRAF3IP3 mediates BRAF-MEK interaction and MEK activation in Golgi
To assess the molecular mechanism by which TRAF3IP3 regulates ERK signaling, we analyzed the upstream signaling events. Consistent with a prior study (Tsukamoto et al., 2008), TCR cross-linking by anti-CD3 plus anti-CD4 stimulated the activation of BRAF but not RAF1 in DP thymocytes (Fig. 8 A). Surprisingly, the TRAF3IP3 deficiency did not affect the catalytic activation of BRAF (Fig. 8 A). This unexpected result prompted us to examine whether TRAF3IP3 regulated the interaction of BRAF with MEK. In response to TCR stimulation, BRAF rapidly bound MEK in WT DP thymocytes; however, this molecular interaction was severely attenuated in the Traf3ip3-KO DP thymocytes (Fig. 8 B). translocation of BRAF to the Golgi, although this latter event was independent of TRAF3IP3 ( Fig. 8 H). Collectively, these results suggest that TRAF3IP3 may recruit MEK to the Golgi, thereby facilitating TCR-stimulated BRAF-MEK interaction and MEK phosphorylation in the Golgi.

The transmembrane domain of TRAF3IP3 is required for its function
We next examined the role of the transmembrane domain of TRAF3IP3 in mediating its subcellular localization and signaling function. We constructed retroviral vectors encoding WT TRAF3IP3 or its mutant lacking the transmembrane domain (TM) fused with CFP ( Fig. 9 A). When stably expressed Traf3ip3-KO cells (Fig. 8 G). Furthermore, in response to TCR/CD28 stimulation, a proportion of BRAF was transferred to the membrane in a TRAF3IP3-independent manner.
MAPK signaling is known to occur in the membrane of intracellular organelles, particularly the Golgi, and the Golgispecific MAPK signaling has been implicated in the regulation of thymocyte positive selection (Daniels et al., 2006;Mayinger, 2011). Interestingly, subcellular fractionation studies readily revealed the association of TRAF3IP3 with the Golgi (Fig. 8 H). Moreover, MEK was located to the Golgi in a TRAF3IP3-dependent manner and became phosphorylated within the Golgi in response to TCR stimulation (Fig. 8 H). The MEK phosphorylation was associated with TCR-stimulated  Traf3ip3-KO BM cells with WT or TM TRAF3IP3 and adoptively transferred the BM cells into Rag1-KO mice. Although TRAF3IP3 and its TM mutant were comparably expressed in the thymocytes of the BM chimeric mice (Fig. 9 D), expression of WT TRAF3IP3, but not TM, rescued the defect in thymocyte development (Fig. 9 E) and ERK phosphorylation (Fig. 9 F). Importantly, the WT TRAF3IP3, in Jurkat T cells (Fig. 9 B), the WT TRAF3IP3 was located in the membrane, suggesting that the CFP tag did not affect its subcellular localization (Fig. 9 C). In contrast to the WT TRAF3IP3, the TM mutant was predominantly in the cytosol, thus confirming the function of the transmembrane domain ( Fig. 9 C). To study the function of the TRAF3IP3 transmembrane domain in thymocytes, we transduced the and a C-terminal transmembrane domain. We found that the transmembrane domain of TRAF3IP3 was indeed required for its association with membrane and localization to the Golgi apparatus. Importantly, the function of TRAF3IP3 in mediating MEK-ERK activation and thymocyte development also relied on its transmembrane domain. These findings shed additional light onto the mechanism by which TRAF3IP3 mediates TCR signaling in thymocytes.
A prior study suggests that overexpressed TRAF3IP3 induces JNK activation in cooperation with TRAF3 (Dadgostar et al., 2003). This function of TRAF3IP3 may involve its association with TRAF3 and recruitment of TRAF3 to detergent-insoluble cellular compartments (Dadgostar et al., 2003). Our preliminary studies failed to detect appreciable binding of TRAF3IP3 with TRAF3 in thymocytes (unpublished data). Furthermore, the TRAF3IP3 deficiency did not affect TCR-stimulated activation of JNK in thymocytes, although it remains possible that TRAF3IP3 is required for JNK activation in other cell types. Nevertheless, because TRAF3 is dispensable for thymocyte development (Xie et al., 2011), it is unlikely that the function of TRAF3IP3 in thymocytes is mediated by TRAF3. Instead, our data suggest a specific role for TRAF3IP3 in mediating MEK-ERK activation and DP thymocyte positive selection.

Mice.
Traf3ip3-targeted mice, Traf3ip3 tm1a(KOMP)Wtsi (in C57BL/6N background), were generated at Knockout Mouse Project (KOMP) by targeting exon 3 (first coding exon) of Traf3ip3 gene using a FRT-LoxP vector (Fig. 1 B). Germline Traf3ip3-KO mice were generated by crossing the Traf3ip3targeted mice with CMV-Cre mice (The Jackson Laboratory, C57BL/6 background). Heterozygous (Traf3ip3 +/ ) mice were bred to generate agematched WT and homozygous Traf3ip3-KO experimental mice. Traf3ip3flox mice were generated by crossing the Traf3ip3-targeted mice with FLP deleter mice (Rosa26-FLPe; 129S4/Sv background; The Jackson Laboratory), and the Traf3ip3-flox mice were further crossed with Cd4-Cre mice (The Jackson Laboratory) to generate Traf3ip3-TKO and WT (Traf3ip3 +/+ Cd4-Cre) mice. Genotyping was performed as indicated in Fig. 1 (C and E) using primers listed in Table S1. B6.SJL mice (expressing the CD45.1 congenic marker), Rag1-KO mice, OT-I and OT-II TCR-transgenic mice, and R26Stop FL MEK1DD (Map2k1*) mice were from the Jackson Laboratory. H-Y TCR transgenic mice were provided by M. Demetriou (University of California, Irvine, Irvine, CA). All of these mouse strains were in C57BL/6 background, except the Traf3ip3-TKO mice, which were in B6/129 mixed background. Mice were maintained in a specific pathogen-free facility, and all animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Texas MD Anderson Cancer Center.
Plasmids, antibodies, and reagents. pcDNA-HA-TRAF3IP3 was created by inserting mouse TRAF3IP3 into the pcDNA3-HA vector; pCLXSN-TRAF3IP3-CFP and pCLXSN-TRAF3IP3TM were generated by inserting the mouse TRAF3IP3 and TRAF3IP3TM (lacking the C-terminal 25 amino acids containing a transmembrane domain), fused with CFP, into the pCLXSN retroviral vector (Naviaux et al., 1996). pCLXSN-CFP was generated by inserting CFP cDNA into pCLXSN, and mouse HA-MEK1 plasmid was purchased from Addgene. Antibodies for -Adaptin but not its TM mutant, located to the Golgi and recruited MEK to the Golgi (Fig. 9 G). Thus, the transmembrane domain of TRAF3IP3 is required for its Golgi localization and mediating compartmentalized MEK activation during thymocyte development.

DISCUSSION
The results described in this paper established TRAF3IP3 as a crucial regulator of thymocyte development. TRAF3IP3 specifically mediated TCR-stimulated activation of MEK and ERK. Consistent with the requirement of the MEK-ERK pathway in thymocyte development, we obtained genetic evidence that the attenuated MEK-ERK signaling in TRAF3IP3-deficient thymocytes critically contributed to their developmental defect.
Our data suggested a role for TRAF3IP3 in mediating the late stages of thymocyte development. Germline ablation of TRAF3IP3 did not affect the DN thymocyte development or the generation of immature DP thymocytes. In fact, the frequency of DP thymocytes was substantially elevated in the Traf3ip3-KO mice, consistent with a block in their progression to the mature SP stage. These results were corroborated by the finding that TRAF3IP3 ablation during DP stage of thymocyte development in the Traf3ip3-TKO mice also caused a defect in SP thymocyte generation. We obtained strong evidence that TRAF3IP3 was involved in positive selection of the DP thymocytes. Based on the analysis of CD69 expression, the TRAF3IP3 deficiency greatly reduced the populations of DP thymocytes that were undergoing, or had completed, positive selection. Similar results were obtained through analysis of another positive selection marker, CD5 (unpublished data). The role of TRAF3IP3 in mediating positive selection was further confirmed by using TCR transgenic mice. This specific function of TRAF3IP3 was correlated with its predominant expression in DP thymocytes.
In thymocytes, BRAF is largely responsible for TCRstimulated MEK-ERK activation and required for thymocyte positive selection (Dillon et al., 2013). However, how BRAF transduces the TCR signal to the activation of downstream kinases has remained elusive. We found that BRAF rapidly moved to Golgi in response to TCR stimulation, suggesting Golgi-specific activation and/or function of this MEK kinase. Although how BRAF is recruited to the Golgi remains to be further studied, our findings suggest a TRAF3IP3independent mechanism. TRAF3IP3 is also dispensable for the catalytic activation of BRAF. In contrast, TRAF3IP3 was required for MEK localization to the Golgi and TCRstimulated MEK-BRAF physical association. It is thus likely that TRAF3IP3 may facilitate the interaction of BRAF with MEK in the Golgi, thereby allowing BRAF to phosphorylate and activate MEK. Indeed, MEK became phosphorylated in the Golgi upon TCR stimulation, which was dependent on TRAF3IP3. Thus, our findings provide an example for how the BRAF-MEK-ERK signaling cascade is activated in a specific subcellular compartment during T cell development. The sequence of TRAF3IP3 predicts a coiled-coil domain followed by staining with secondary Alexa Fluor 488 or 647 antibodies (Molecular Probes) for 30 min. Slides were mounted in antifade reagent with DAPI. Pictures were taken with an SP5 RS confocal microscope (Leica) and analyzed by SlideBook 5.0 software. Statistical analysis. Statistical analysis was performed using Prism software (GraphPad Software). Two-tailed unpaired Student's t tests were performed, p-values <0.05 were considered significant, and the level of significance was indicated as follows: *, P < 0.05; **, P < 0.01. In the animal experiments, four mice are required for each group based on the calculation to achieve a 2.3-fold change (effect size) in two-tailed Student's t test with 90% power and a significance level of 5%.
Online supplemental material. Table S1 list the primers used for genotyping PCR. Online supplemental material is available at http://www.jem .org/cgi/content/full/jem.20150110/DC1.