MIP-T3, a Novel Protein Linking Tumor Necrosis Factor Receptor-associated Factor 3 to the Microtubule Network*

In this study, we report the identification of a novel tumor necrosis factor receptor-associated factor 3 (TRAF3)-interacting protein designated MIP-T3. MIP-T3 is a 83-kDa protein with no significant homology to known mammalian proteins. MIP-T3 mRNA and TRAF3 mRNA are ubiquitously expressed, and TRAF3 is the only TRAF protein to interact with MIP-T3. The MIP-T3-TRAF3 interaction requires the coiled-coil TRAF-N domain of TRAF3. To our knowledge, this is the first case of a TRAF-binding protein that interacts with a single member of the TRAF family specifically through a TRAF-N coiled-coil domain. MIP-T3 binds to Taxol-stabilized microtubules and to tubulin in vitro, and MIP-T3 recruits TRAF3 to microtubules when both proteins are overexpressed in HeLa cells. In a 293 cell line stably expressing CD40, TRAF3 is released from the TRAF3·MIP-T3 complex and recruited to the CD40 receptor upon CD40 ligand stimulation. MIP-T3 may provide a novel mechanism in sequestering TRAF3 to the cytoskeletal network.


Lei Ling ‡ and David V. Goeddel
From Tularik Inc., South San Francisco, California 94080 In this study, we report the identification of a novel tumor necrosis factor receptor-associated factor 3 (TRAF3)-interacting protein designated MIP-T3. MIP-T3 is a 83-kDa protein with no significant homology to known mammalian proteins. MIP-T3 mRNA and TRAF3 mRNA are ubiquitously expressed, and TRAF3 is the only TRAF protein to interact with MIP-T3. The MIP-T3-TRAF3 interaction requires the coiled-coil TRAF-N domain of TRAF3. To our knowledge, this is the first case of a TRAF-binding protein that interacts with a single member of the TRAF family specifically through a TRAF-N coiled-coil domain. MIP-T3 binds to Taxol-stabilized microtubules and to tubulin in vitro, and MIP-T3 recruits TRAF3 to microtubules when both proteins are overexpressed in HeLa cells. In a 293 cell line stably expressing CD40, TRAF3 is released from the TRAF3⅐MIP-T3 complex and recruited to the CD40 receptor upon CD40 ligand stimulation. MIP-T3 may provide a novel mechanism in sequestering TRAF3 to the cytoskeletal network.
Overexpression of TRAF2, TRAF5, or TRAF6 activates NF-B, and truncated versions of TRAF2, TRAF5, and TRAF6 lacking zinc binding domains act as dominant negative inhibitors of NF-B activation mediated by various receptors, suggesting that these TRAFs are common mediators for NF-B activation (9,10,12,13,29,32). On the other hand, TRAF1, TRAF3, and TRAF4 do not activate NF-B when overexpressed (16). TRAF2, TRAF5, and TRAF6 also activate c-Jun N-terminal kinase when overexpressed (16,17). Additional data on the physiological roles of four TRAF family members have been determined by gene targeting experiments in mice (18 -23). TRAF2- (18), TRAF3- (21), and TRAF6deficient (23) mice appear normal at birth but become progressively runted and die prematurely. TRAF2-deficient mice have elevated levels of serum TNF. Ex vivo assays demonstrated that TRAF2-/-embryonic fibroblasts are highly sensitive to TNF-induced cell death and that in the absence of TRAF2, TNF-mediated c-Jun N-terminal kinase activation is greatly reduced. TRAF2 deficiency results in partial inhibition of TNF-induced NF-B activation, and complete inhibition of CD40-induced NF-B activation (18,19). Recent studies on TRAF6 -/-mice revealed that TRAF6 is required for interleukin 1, CD40, and lipopolysaccharide-induced NF-B activation (23). Moreover, TRAF6-deficient mice are osteopetrotic, with defects in bone remodeling and tooth eruption due to impaired osteoclast function (23). Unlike TRAF2-, TRAF3-, and TRAF6deficient mice, TRAF5-/-mice are healthy through 24 weeks of age, but the CD40 and CD27 signaling pathways are impaired in these mice (22). Collectively, these results suggested that TRAF2, TRAF5, and TRAF6 could act redundantly or specifically in particular signaling cascades.
TRAF3 was originally identified as a molecule that binds the cytoplasmic domains of the TNFR family member CD40 and the Epstein-Barr virus latent membrane protein LMP1 (4 -7). Subsequently, it has been shown to interact with the cytoplasmic tails of the TNFR family members lymphotoxin ␤-receptor (LT␤R), CD27, CD30, OX40, HEPES virus entry mediator, and receptor activator of NF-B (24 -32). The interaction of TRAF3 with both CD40 and LT␤R has been shown to be ligand-dependent and to occur in nontransfected cells (33,34). However, deletion of TRAF3 by gene targeting does not seem to affect either CD40-induced B cell proliferation or CD23 up-regulation in mice (21), nor do TRAF3-deficient mice have defective lymph node genesis as seen in LT␤R-knockout mice (21). However, reconstitution of mice with TRAF3-/-fetal liver cells revealed a requirement for TRAF3 in T cell-dependent immune responses (21).
To gain more insights about the physiological roles of TRAF3, we performed yeast two-hybrid cloning to search for * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF230877.
TRAF3-interacting proteins. We identified a novel protein, MIP-T3, that is endogenously associated with TRAF3 and that appears to provide a link between TRAF3 and the microtubule network.
Expression Vectors-Mammalian expression vectors encoding TRAFs 1-6 have been described (16). MIP-T3 was expressed as N-terminally tagged Flag or Myc fusion proteins using the pRK vector. Deletion mutants of MIP-T3 and TRAF3 were generated by polymerase chain reaction.
Yeast Two-hybrid Cloning-DNA encoding full-length TRAF3 was used as a bait in the MATCHMAKER LexA two-hybrid system (CLON-TECH). The two-hybrid screening was performed according to the manufacturer's instructions except that a pretransformed HeLa cDNA library (generously provided by Dr. Hsing-Jien Kung) was used. Positive yeast clones were selected by prototrophy for leucine and expression of ␤-galactosidase. Yeast DNA was recovered and transformed into Escherichia coli. Plasmids containing cDNA clones were identified by restriction mapping and further characterized by DNA sequencing.
cDNA Cloning and Northern Blot Hybridization-The 1-kb cDNA insert from the partial two-hybrid MIP-T3 clone was used as a probe to screen human HeLa cDNA library in Zap (provided by Dr. Z. Cao) by standard methods. DNA sequencing was performed on an Applied Biosystems automated DNA sequencer. Northern blot analysis of the human multiple tissue blot (CLONTECH) was performed according to the instructions of the manufacturer, using a 1-kb EcoRI-EcoRI fragment from the MIP-T3 cDNA as probe.
Transfection, Immunoprecipitation, and Immunoblotting Analyses-293 cells or HeLa cells were transiently transfected with expression plasmids using the calcium phosphate method. 24 -36 h later, cells were washed with cold phosphate-buffered saline and lysed in Lysis Buffer A containing 20 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and the Complete TM protease inhibitors (Roche). Cell lysates were cleared and incubated for 2-4 h at 4°C with anti-Flag M2 antibody or control mouse IgG and Gamma-Bind G Plus-Sepharose beads (Amersham Pharmacia Biotech). The Sepharose beads were washed extensively with lysis buffer. Samples were analyzed by SDS-PAGE and transferred to Immobilon polyvinylidene difluoride membrane (Millipore). Immunoblotting analyses were performed with various polyclonal antibodies and detected by alkaline phosphatase-conjugated goat-anti-rabbit IgG (Bio-Rad), horseradish peroxidase-coupled goat-anti-rabbit IgG (Amersham Pharmacia Biotech), or horseradish peroxidase-coupled protein A (Amersham Pharmacia Biotech).
For GST pull-down assays, the GST fusion proteins (GST-TRAF3 and GST-MIP-T3 [aa 324 -625]) were expressed in bacteria after isopropyl-1-thio-␤-D-galactopyranoside induction using the pGEX vector (Amersham Pharmacia Biotech) and purified on glutathione beads (Amersham Pharmacia Biotech). Full-length and deletion mutants of TRAF3 and MIP-T3 were in vitro translated using the TNT TM SP6coupled reticulocyte lysate system (Promega). 10 l of in vitro translation products were diluted to 400 l with Lysis Buffer A and incubated with 2 g of GST or GST fusion protein immobilized on glutathione beads. The samples were then washed extensively, fractionated by SDS-PAGE, and exposed to x-ray films.
For detection of the endogenous interaction of TRAF3 with MIP-T3, untransfected HeLa cells, 293 cells, or 293.CD40 cells (a 293 cell line stably expressing Flag-tagged CD40, generously provided by Dr. Ralf Schwandner) were not stimulated or stimulated with a 250g/ml-membrane preparation of CD40 ligand (35) for various times. The cells were then lysed in Lysis Buffer A. Immunoprecipitation was carried out using affinity-purified rabbit-anti-MIP-T3 antibody, and the immunoprecipitates were immunoblotted with anti-TRAF3 antibodies.
Indirect Immunofluorescence Microscopy-HeLa cells were tran-siently transfected with various plasmids. When indicated, 8 h after transfection, cells were treated with 5 g/ml Taxol or 400 ng/ml nocodazole overnight. 24 h after transfection, cells were fixed with 3.7% formaldehyde in phosphate-buffered saline, permeabilized with 0.1% Triton X-100, and blocked with 10% goat serum. Then cells were incubated with various combinations of anti-Myc, anti-Flag, and/or anti-tubulin antibodies, followed by FITC-conjugated anti-rabbit secondary antibody and/or rhodamine-conjugated anti-mouse secondary antibody, and examined under a Zeiss microscope at ϫ 400 magnification. Cell images were captured with the Sensys CCD camera system (Photometrics) and analyzed by Image-Pro Plus (Media Cybernetics) and Adobe Photoshop.
Microtubule-associated Protein (MAP) Spin-down Assays-MIP-T3 proteins were in vitro translated using the SP6 TNT TM -coupled reticulocyte lysate systems (Promega). Taxol-stabilized microtubules were assembled from dimeric tubulin (Cytoskeleton) in G-PEM buffer (80 mM Pipes (pH 6.8), 1 mM MgCl 2 , 1 mM EGTA, and 1 mM GTP). The in vitro translated proteins were incubated with microtubules at room temperature for 20 min, loaded onto cushion buffer (PEM buffer plus 40% glycerol) and centrifuged at 100,000 ϫ g for 40 min at room temperature. The pellets were washed with G-PEM and analyzed by SDS-PAGE.

Isolation of cDNA Clones Encoding TRAF3-interacting Pro-
teins-To increase our understanding of the function of TRAF3, we performed yeast two-hybrid interaction cloning to screen for proteins that associate directly with TRAF3. An expression vector in which full-length TRAF3 was fused to the LexA DNA binding domain was used as a bait to screen a HeLa cDNA library (36). From approximately 10 million transformants, 10 independent positive clones were obtained, as determined by activation of Leu and LacZ reporter genes. One of these clones encodes I-TRAF/TANK, a protein previously shown to be associated with various TRAFs (37,38). Three of these clones were independent isolates encoding portions of a novel protein. The longest insert from these three clones contains an open reading frame encoding 302 amino acids. The interaction between TRAF3 and this 302 amino acid protein was verified by retransformation into yeast cells (data not shown).
We screened a HeLa ZAP cDNA library using the partial cDNA as probe. DNA sequence analysis of the positive clones revealed an open reading frame predicted to encode a protein of 625 amino acids that we have designated MIP-T3, for microtubule-interacting protein that associates with TRAF3 (Fig. 1). Data base searches utilizing the BLAST and FASTA programs failed to identify any mammalian proteins having significant sequence similarity to MIP-T3. However, a Caenorhabditis elegans protein (GenBank TM accession number U49945) of unknown function shares an overall 22% identity and 29% similarity to MIP-T3. The C-terminal 100 amino acids of MIP-T3 form a coiled-coil domain (aa 525-625).
Northern blot analysis indicated that MIP-T3 mRNA was expressed in all human tissues examined ( Fig. 2A). This result is consistent with MIP-T3 involvement in TRAF3 signal transduction, as TRAF3 is also expressed ubiquitously (6,39). Two different sizes (4.4 and 2.4 kb) of MIP-T3 transcripts were visualized in most tissues on the Northern blots. The two transcripts encode the same open reading frame with the longer transcript having an extra 2 kb of 3Ј untranslated region, as revealed from the sequences of MIP-T3 clones from the HeLa Zap cDNA library. In addition, testis contains an additional MIP-T3 transcript of 2.7 kb.
Polyclonal antibody against MIP-T3 was generated by immunizing rabbits with a 35-mer peptide, KKILETKKDYEK-LQQSPKPGEKERSLFESAWKKEK, derived from amino acids 476 -510 of MIP-T3. Rabbit anti-MIP-T3 antiserum specifically recognized a protein of approximately 83 kDa in nontransfected 293, HeLa, and SW480 cells by immunoprecipitation followed by immunoblotting with the same antibody (Fig. 2B), which is the same size as the MIP-T3 expressed in transiently transfected 293 cells.
MIP-T3 Specifically Interacts with TRAF3 in Vitro and in Vivo-We performed in vitro GST pull-down assays to confirm the interaction between MIP-T3 and TRAF3 observed in the two-hybrid system. All six TRAFs were 35 S-labeled by in vitro translation using SP6 RNA polymerase. Equal amounts of TRAFs were incubated with GST or GST-MIP-T3 (aa 324 -625) protein immobilized on glutathione beads. As shown in Fig. 3A, MIP-T3 strongly associated with TRAF3 but not with TRAF1, TRAF2, TRAF4, or TRAF5. MIP-T3 also very weakly associated with TRAF6. The interaction of MIP-T3 with TRAF3 was further analyzed in mammalian cell coimmunoprecipitation assays. The full-length MIP-T3 construct containing an Nterminal Myc epitope tag was transiently coexpressed in 293 cells with Flag epitope-tagged TRAFs. Cell lysates were immunoprecipitated using a monoclonal antibody against the Flag epitope or control mouse IgG, and coprecipitating MIP-T3 was detected by immunoblotting analyses with anti-Myc polyclonal antibody (Fig. 3B). In this assay, MIP-T3 specifically coprecipitates TRAF3 but not TRAF1, TRAF2, TRAF4, TRAF5, or TRAF6, consistent with the results of the in vitro GST pulldown assay.
The Coiled-coil Regions in TRAF3 and MIP-T3 Contribute to Their Interaction-TRAF3 contains a N-terminal ring finger domain followed by several zinc fingers (4,38). The C-terminal half of TRAF3 is the TRAF domain, which is conserved among all six members of the TRAF family. The TRAF domain of TRAF3 can be further subdivided into the TRAF-N (aa 264 -415) and TRAF-C (aa 416 -568) domains (12,38). The TRAF-N domain of TRAF3 contains an extended coiled-coil region (4,12,38). To determine which regions of TRAF3 contribute to MIP-T3 binding, various 35 S-labeled TRAF3 deletion mutants were assayed for association with a GST-MIP-T3 (aa 324 -625) fusion protein (Fig. 4A). The region containing the coiled-coil TRAF-N domain of TRAF3 (aa 267-376) is sufficient for bind-ing to MIP-T3. Similar results were observed in 293 cell cotransfection experiments (Fig. 4B). Only TRAF3 deletion mutants (aa 1-376 and 267-568) containing the coiled-coil TRAF-N region were able to retain MIP-T3 binding activity, whereas TRAF3 deletion mutants (aa 1-112, 1-266, and 390 -568) corresponding to other domains of TRAF3 were not able to bind MIP-T3. These binding results are summarized in Fig. 4C.
Similarly, to determine which domains of MIP-T3 contribute to the interaction with TRAF3, various MIP-T3 deletion mutants were generated. In the GST pull-down assays, the coiledcoil region of MIP-T3 (aa 525-625) is sufficient for binding to TRAF3 (Fig. 5A). Similar results were observed in 293 cotransfection experiments (Fig. 5B). In summary, it appears that the coiled-coil regions of MIP-T3 and TRAF3 contribute to their association (Figs. 5C and 4C, respectively).
MIP-T3 and TRAF3 Interact Endogenously-Protein-protein interactions that occur when the proteins are artificially over-expressed may not exist in untransfected cells. We therefore examined the nontransfected human cell lines 293 and HeLa to determine whether endogenous MIP-T3 and TRAF3 interact under physiological conditions. Cell lysates were immunoprecipitated with an anti-MIP-T3 rabbit polyclonal antibody. In both cell types, coprecipitating TRAF3 was readily detected by immunoblotting with anti-TRAF3 polyclonal antibody (Fig. 6).
Association of the MIP-T3⅐TRAF3 Complex with Microtubular Structures-To gain insight into the functional importance of MIP-T3 and TRAF3, we studied their subcellular localization by immunofluorescence microscopy. HeLa cells were transiently transfected with Myc-tagged MIP-T3 or Flag-tagged TRAF3. These epitope tags enabled us to visualize the expressed proteins by immunofluorescence using the corresponding epitope-specific affinity-purified antibodies. 24 h after transfection, the cells were fixed, permeabilized, and then incubated with anti-Myc or anti-Flag primary antibodies followed by fluorescently labeled secondary antibodies. Counter staining with DAPI was included to visualize the nucleus (data not shown). When expressed separately, both MIP-T3 and TRAF3 had a cytosolic localization (Fig. 7, A and B); however, when MIP-T3 and TRAF3 were co-expressed, both proteins localized to cytoskeletal structures (Fig. 7C). Pretreating cells with 0.2% Triton before fixation (40) showed the same cytoskeleton localization results (data not shown). Several properties indicate that this cytoskeletal structure is very likely microtubules. First, treatment with Taxol (a microtubule stabilizing drug) leads to the formation of organized bundles around the nucleus (41). Second, treatment with nocodazole (a microtubule destabilizing drug) destroyed this structure (41) (Fig. 7C). Indeed, simultaneously staining of these cells with anti-Myc (to visualize MIP-T3) and anti-tubulin antibodies revealed the co-localization of MIP-T3 and tubulin (Fig. 7D). Similarly, staining of these cells with anti-Flag (to visualize TRAF3) and anti-tubulin antibodies revealed the co-localization of TRAF3 and tubulin (data not shown). Therefore, MIP-T3 and TRAF3 are co-localized to microtubule structure when co-expressed. This co-localization was confirmed by confocal analyses (data not shown). It is noteworthy that after treatment of cells with nocodazole and complete microtubule depolymerization, MIP-T3 and TRAF3 remain associated with a patchy staining pattern (Fig. 7, C and D), suggesting that MIP-T3 and TRAF3 are able to associate with other intracellular structures and with microtubules (42).  Next, we co-expressed Myc-tagged MIP-T3 with six different Flag-tagged TRAF proteins in HeLa cells. The transfected cells were simultaneously stained with anti-Myc antibody to visualize MIP-T3 (Fig. 8) and with anti-Flag antibody to visualize co-transfected TRAFs. MIP-T3 localized to the microtubules only when co-expressed with TRAF3 (Fig. 8). Similarly, TRAF3 was the only TRAF protein to localize to microtubules when co-expressed with MIP-T3 (data not shown).
MIP-T3 Binds to Taxol-stabilized Microtubules and to Tubulin-To examine the ability of MIP-T3 to bind to microtubules in vitro, we utilized the MAP spin-down assay (43,44). Taxol-stabilized microtubules were incubated with in vitro translated MIP-T3. The microtubule pellets were collected by centrifugation, washed, and analyzed for the presence of MIP-T3 by autoradiography (Fig. 9A). MIP-T3 was able to bind polymerized microtubules (Fig. 9A, lane 1), whereas no microtubule binding was observed for negative control BSA protein (data not shown). No MIP-T3 protein was pelleted in the absence of microtubules.
To determine whether the MIP-T3 protein contains a specific domain that might be responsible for microtubule binding, various MIP-T3 deletion mutants were used. These mutants HeLa cells were co-transfected with Flagtagged TRAF3 and Myc-tagged MIP-T3 and stimulated with 5 g/ml Taxol or 400 ng/ml nocodazole as indicated. Cells were probed with both anti-Flag and anti-Myc primary antibodies, followed by rhodamine-and FITC-conjugated secondary antibodies, respectively. TRAF3 was visualized with rhodamine and is shown in the left panels. MIP-T3 was visualized with FITC and is shown in the middle panels. The superimposed pictures are shown in the right panels. Yellow indicates co-localization. D, localization of TRAF3 and MIP-T3 to microtubules when co-expressed. Procedures were the same as in C except that cells were probed with both anti-Myc and anti-tubulin primary antibodies followed by FITC-and rhodamine-conjugated secondary antibodies, respectively. MIP-T3 was visualized with FITC and is shown in the left panels, and tubulin was visualized with rhodamine and is shown in the middle panels. The superimposed pictures are shown in the right panels. Yellow indicates co-localization.
were synthesized by in vitro translation and subjected to the MAP spin-down assay. As shown in Fig. 9A, deletion of Cterminal coiled-coil region of MIP-T3 does not affect the microtubule binding ability of MIP-T3. However, deletion of the N-terminal 250 amino acids significantly reduced the binding of MIP-T3 to microtubules. These results demonstrate that MIP-T3 utilizes different regions for binding to TRAF3 and microtubules.
Similarly, immobilized GST-MIP-T3 fusion proteins were incubated with purified tubulin. After extensive washing, the tubulin bound on GST fusion protein beads was detected by immunoblotting with anti-tubulin antibody (Fig. 9B). GST protein alone does not bind tubulin. GST-MIP-T3 (full-length) protein, as well as GST-MIP-T3 (aa 1-525) and GST-MIP-T3 (aa 1-425) fusion proteins bind tubulin efficiently. Deletion of the N-terminal 323 amino acids of MIP-T3 abolishes its tubulin binding ability, which is consistent with the MAP spin-down assay results. The microtubule binding properties of MIP-T3 are summarized in Fig. 9C.
CD40 Ligand Induces Dissociation of TRAF3 from the TRAF3⅐MIP-T3 Complex-To examine the effect of cytokines on the complex formation of MIP-T3 with TRAF3, we used a 293.CD40 cell line that stably expresses Flag-tagged CD40. Following stimulation with a membrane preparation of CD40 ligand for various times, 293.CD40 cells were lysed, and extracts were immunoprecipitated with an anti-MIP-T3 rabbit polyclonal antibody or with an anti-Flag monoclonal antibody. The coprecipitating TRAF3 was detected by immunoblotting with an anti-TRAF3 monoclonal antibody. These experiments showed that TRAF3 dissociates from the endogenous TRAF3⅐MIP-T3 complex and is recruited to the CD40 receptor complex following CD40 ligand stimulation in a time-dependent manner (Fig. 10).

DISCUSSION
In this study, we report the identification of a novel TRAF3interacting protein, MIP-T3. MIP-T3 is a 83-kDa protein with no significant homology to known mammalian proteins. Like TRAF3, MIP-T3 is also ubiquitously expressed. Interestingly, TRAF3 is the only TRAF protein that interacts with MIP-T3, and this interaction requires the coiled-coil TRAF-N domain of TRAF3. To our knowledge, this is the first case of a TRAFbinding protein that interacts with a single member of the TRAF family specifically through a coiled-coil TRAF-N domain. It has been shown that the coiled-coil domain of TRAF3 is also important for efficient recruitment of TRAF3 to the LT␤R and for TRAF3 self-association (45). We evaluated the ability of MIP-T3 to activate NF-B, a known TRAF-mediated pathway. Overexpression of MIP-T3 alone or in combination with TRAF3 failed to activate NF-B (data not shown). This result is consistent with data showing that TRAF3 is not required for the activation of NF-B-dependent pathways by the CD40 ligand in TRAF3-deficient mice (21).
An interesting feature of MIP-T3 was revealed by immunofluorescence microscopy studies. MIP-T3 and TRAF3 are cytosolic proteins when expressed separately. However, when MIP-T3 and TRAF3 are co-expressed, they both localize to microtubular structures (Figs. 7 and 8). Moreover, MIP-T3 binds to Taxol-stabilized microtubules and to tubulin in vitro. It can be postulated that the interaction between MIP-T3 and TRAF3 further facilitates the microtubule binding ability of MIP-T3. We observed that when cells express a high level of MIP-T3 (for example, when high levels of DNA are transfected or when cells are stained 40 h after transfection), MIP-T3 itself can localize to microtubule even in the absence of TRAF3 (data not shown). When MIP-T3 is expressed at low levels, it is mainly found in the cytosol, with co-expression of TRAF3 being required to bring both proteins to microtubules. However, it remains to be investigated whether co-expression of MIP-T3 and TRAF3 increases the stability of microtubules. It would also be interesting to determine whether the MIP-T3-TRAF3 interaction is regulated by phosphorylation, as the microtubule-association of many MAPs is regulated by phosphorylation (46 -48). We also noticed that after treatment of cells with nocodazole and complete microtubule depolymerization, MIP-T3 and TRAF3 remain associated with a patchy staining pattern (Fig. 7, C and D), suggesting that MIP-T3 and TRAF3 are able to associate with other intracellular structures and with microtubules (42).
The microtubule binding domain of MIP-T3 was localized to the N-terminal region, between amino acids 51 and 250. This region is highly charged and rich in lysine and glutamic acid residues. The predominance of lysine and glutamic acid residues is also a typical feature of the tubulin binding region in MAP-1B, which contains reiterated KKE motifs (49). These motifs appear to be important for the interaction of MAP-1B with microtubules (49). Because there is a KKE motif around amino acid 243 of MIP-T3, it seems possible that the interaction of MIP-T3 with tubulin may occur by a similar mechanism.
Interestingly, the MIP-T3⅐TRAF3 complex exists in nontransfected cells. This complex can be dissociated upon CD40 ligand stimulation in 293.CD40 cells, whereas TRAF3 is recruited to the CD40 receptor. The sequestering of TRAF3 by MIP-T3 is reminiscent of the regulation of Smads (50). In transforming growth factor ␤ pathway, microtubules serve as a cytoplasmic sequestering network for Smads in unstimulated cells. Transforming growth factor ␤ triggers dissociation of Smads from microtubule, phosphorylation, and nuclear localization of Smad2 and Smad3, with consequent activation of transcription inside the nucleus (50).
Our experiments provide in vitro and in vivo evidence for TRAF3⅐MIP-T3/microtubule association and evidence that TRAF3⅐MIP-T3 association can be further regulated by activation of cytokine receptors such as CD40. It has been demonstrated that tubulin and microtubules are capable of influencing cellular signaling through direct interaction with a variety of signaling molecules (51)(52)(53)(54). Therefore, it is possible that through binding to MIP-T3, tubulin or microtubules may regulate the cellular localization and/or functions of MIP-T3 and TRAF3. Additionally, tubulin is known to associate with a variety of cellular membranes, including the plasma membrane, endosomes, microsomes, and various organelles (51). Thus, the potential exists that microtubules could be responsible for directing MIP-T3 and TRAF3 to defined membrane microdomains in the cell. Finally, it is possible that such interactions may be dynamically regulated by stimuli in coordination with dynamic functions of the microtubule cytoskeleton during processes such as cell growth and differentiation.
In summary, we have identified a novel protein, MIP-T3, that interacts with TRAF3 and with microtubular structures. CD40 ligand stimulation induces the dissociation of the TRAF3⅐MIP-T3 complex. This signaling cascade could lead to the reorganization of microtubular cytoskeleton network, which direct cells for adhesion, movement, secretion, and other responses. Further investigations will be necessary to identify the precise roles of MIP-T3 during its association with TRAF3 and microtubules in cytokine signaling pathways.