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Jamip1 (Marlin-1) Defines a Family of Proteins Interacting with Janus Kinases and Microtubules

2004; Elsevier BV; Volume: 279; Issue: 41 Linguagem: Inglês

10.1074/jbc.m401915200

1083-351X

Corinna Steindler, Zhi Li, Michèle Algarté, Andrés Alcover, Valentina Libri, Josiane Ragimbeau, Sandra Pellegrini,

Microtubule and mitosis dynamics

Jamip1 (Jak and microtubule interacting protein), an alias of Marlin-1, was identified for its ability to bind to the FERM (band 4.1 ezrin/radixin/moesin) homology domain of Tyk2, a member of the Janus kinase (Jak) family of non-receptor tyrosine kinases that are central elements of cytokine signaling cascades. Jamip1 belongs to a family of three genes conserved in vertebrates and is predominantly expressed in neural tissues and lymphoid organs. Jamip proteins lack known domains and are extremely rich in predicted coiled coils that mediate dimerization. In our initial characterization of Jamip1 (73 kDa), we found that it comprises an N-terminal region that targets the protein to microtubule polymers and, when overexpressed in fibroblasts, profoundly perturbs the microtubule network, inducing the formation of tight and stable bundles. Jamip1 was shown to associate with two Jak family members, Tyk2 and Jak1, in Jurkat T cells via its C-terminal region. The restricted expression of Jamip1 and its ability to associate to and modify microtubule polymers suggest a specialized function of these proteins in dynamic processes, e.g. cell polarization, segregation of signaling complexes, and vesicle traffic, some of which may involve Jak tyrosine kinases. Jamip1 (Jak and microtubule interacting protein), an alias of Marlin-1, was identified for its ability to bind to the FERM (band 4.1 ezrin/radixin/moesin) homology domain of Tyk2, a member of the Janus kinase (Jak) family of non-receptor tyrosine kinases that are central elements of cytokine signaling cascades. Jamip1 belongs to a family of three genes conserved in vertebrates and is predominantly expressed in neural tissues and lymphoid organs. Jamip proteins lack known domains and are extremely rich in predicted coiled coils that mediate dimerization. In our initial characterization of Jamip1 (73 kDa), we found that it comprises an N-terminal region that targets the protein to microtubule polymers and, when overexpressed in fibroblasts, profoundly perturbs the microtubule network, inducing the formation of tight and stable bundles. Jamip1 was shown to associate with two Jak family members, Tyk2 and Jak1, in Jurkat T cells via its C-terminal region. The restricted expression of Jamip1 and its ability to associate to and modify microtubule polymers suggest a specialized function of these proteins in dynamic processes, e.g. cell polarization, segregation of signaling complexes, and vesicle traffic, some of which may involve Jak tyrosine kinases. The four Jak 1The abbreviations used are: Jak, Janus kinase; Jamip, Jak and microtubule interacting protein; aa, amino acid(s); Ab, antibody; mAb, monoclonal antibody; C-ter, C-terminal region; N-ter, N-terminal region; FERM, band 4.1 ezrin/radixin/moesin; GABAB, γ-aminobutyric acid, type B; GST, glutathione S-transferase; HA, hemagglutinin; IFN, interferon; MES, 4-morpholineethanesulfonic acid; Pipes, 1,4-piperazinediethanesulfonic acid. proteins (Tyk2 and Jak1-3) are 125-140 kDa non-receptor tyrosine kinases that were first identified in mammalian cells for their involvement in signaling via a distinct family of plasma membrane complexes, i.e. the cytokine receptors. These receptors bind cytokines with a common α-helical bundled folding and utilize Jak proteins as their catalytic moieties in a way that recalls the functioning of receptor tyrosine kinases (1Yeh T.C. Pellegrini S. Cell. Mol. Life Sci. 1999; 55: 1523-1534Crossref PubMed Scopus (112) Google Scholar). Since the discovery of the Jak proteins a decade ago much work has been done, ranging from the study of their structure/function organization, post-translational modifications, and subcellular localization to the analysis of knock-out animals and the identification of mutations and translocations responsible for immune deficiency or cancer (2Aringer M. Cheng A. Nelson J.W. Chen M. Sudarshan C. Zhou Y.J. O'Shea J.J. Life Sci. 1999; 64: 2173-2186Crossref PubMed Scopus (64) Google Scholar, 3Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene. 2002; 285: 1-24Crossref PubMed Scopus (914) Google Scholar). These studies have validated the essential role of Jak enzymes in cytokine signaling and helped in understanding their specificity of action. Jak proteins contain an N-terminal FERM (band 4.1 ezrin/radixin/moesin) homology domain, a putative Src homology 2 domain, a regulatory kinase-like domain, and a tyrosine kinase domain activated by tyrosine phosphorylation (1Yeh T.C. Pellegrini S. Cell. Mol. Life Sci. 1999; 55: 1523-1534Crossref PubMed Scopus (112) Google Scholar). Although structure/function analyses suggest a high flexibility and communication among the various parts of the molecule, no crystallographic data are yet available for any of these modular domains. The three-dimensional structures of the moesin and radixin FERM domains were shown to comprise three lobes with similarities to ubiquitin, the acyl-CoA-binding protein, and pleckstrin homology/phosphotyrosine binding-like domains (4Hamada K. Shimizu T. Matsui T. Tsukita S. Hakoshima T. EMBO J. 2000; 19: 4449-4462Crossref PubMed Scopus (317) Google Scholar, 5Pearson M.A. Reczek D. Bretscher A. Karplus P.A. Cell. 2000; 101: 259-270Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). In Jak proteins, the FERM domain is involved in an ill-defined, non-covalent interaction with the membrane-proximal region of cytokine receptors, and, as proposed for the ezrin/radixin/moesin proteins, it may act as a compact architectural unit with multiple binding surfaces. Jaks were reported to bind to a variety of signaling proteins such as SHP1/2, Syp, PP2A, PI3K, Fyn, Yes, Shc, Vav, and Cbl (1Yeh T.C. Pellegrini S. Cell. Mol. Life Sci. 1999; 55: 1523-1534Crossref PubMed Scopus (112) Google Scholar). Other Jak interactors were identified by yeast two-hybrid screens, namely Socs1 (6Endo T.A. Masuhara M. Yokouchi M. Suzuki R. Sakamoto H. Mitsui K. Matsumoto A. Tanimura S. Ohtsubo M. Misawa H. Miyazaki T. Leonor N. Taniguchi T. Fujita T. Kanakura Y. Komiya S. Yoshimura A. Nature. 1997; 387: 921-924Crossref PubMed Scopus (1234) Google Scholar), Stat5 (7Fujitani Y. Hibi M. Fukada T. Takahashi-Tezuka M. Yoshida H. Yamaguchi T. Sugiyama K. Yamanaka Y. Nakajima K. Hirano T. Oncogene. 1997; 14: 751-761Crossref PubMed Scopus (141) Google Scholar), and JBP1/PRMT5, a protein methyltransferase involved in growth control (8Pollack B.P. Kotenko S.V. He W. Izotova L.S. Barnoski B.L. Pestka S. J. Biol. Chem. 1999; 274: 31531-31542Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Some of these complexes were shown to form in a phosphorylation-dependent manner. Recent findings suggest that the binding of Jaks to cytokine receptors may occur early during biosynthesis at the level of endoplasmic reticulum or Golgi membranes, as Jaks were found to potentiate the maturation of at least some receptors (9Huang L.J. Constantinescu S.N. Lodish H.F. Mol. Cell. 2001; 8: 1327-1338Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 10Radtke S. Hermanns H.M. Haan C. Schmitz-Van De Leur H. Gascan H. Heinrich P.C. Behrmann I. J. Biol. Chem. 2002; 277: 11297-11305Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). One family member, Tyk2, was found to stabilize a cognate receptor at the plasma membrane by reducing its rate of internalization (11Ragimbeau J. Dondi E. Alcover A. Eid P. Uze G. Pellegrini S. EMBO J. 2003; 22: 537-547Crossref PubMed Scopus (166) Google Scholar). Another family member, Jak2, was identified as a constituent of the transitional endoplasmic reticulum (12Lavoie C. Chevet E. Roy L. Tonks N.K. Fazel A. Posner B.I. Paiement J. Bergeron J.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13637-13642Crossref PubMed Scopus (81) Google Scholar). Thus, the possibility is emerging that Jaks may not simply act as catalytic moieties of cytokine receptors but may be engaged in interactions with cellular constituents that affect protein assembly and transport processes. Based on these premises, we set out to identify new interactors of Tyk2 using its isolated FERM domain as bait in a two-hybrid screen of a Jurkat T cell line cDNA library. Here, we report on the identification and initial characterization of a protein, which we have designated Jamip1 (acronym of Jak and microtubule interacting protein), that defines a new class of proteins rich in coiled coils and associated with the microtubule network. Our in vivo analyses of wild type and mutated forms of Jamip1 suggest that the protein comprises at least two distinct functional domains, an N-terminal microtubule binding region and a C-terminal regulatory region that can associate with the FERM homology domains of Tyk2 and Jak1. These results, together with the restricted expression of Jamip1 and its paralogue, Jamip2, in neuronal and lymphoid tissues suggest a specialized function of these proteins in processes involving Jak kinases and the dynamic microtubule network. Yeast Two-hybrid Screen—The FERM domain of human Tyk2 (aa 1-451) was cloned in pGBT9 and used as bait in the HF7 yeast strain transformed with a human Jurkat cDNA library in the pACT2 vector (Clontech). 106 transformants were plated on medium lacking histidine. Colonies were then tested for β-galactosidase activity. Transformants were cured of the bait plasmid and re-tested for specific interaction. Yeast plasmid DNA was isolated, rescued into Escherichia coli XL-1, re-transformed into the bait-containing yeast strain, and assayed again. Plasmid Constructs—The full-length Jamip1 coding sequence was obtained by reverse PCR using as template human brain cDNA (Clontech) and cloned in pcDNA3.1/V5-His/neo (Invitrogen) and p3×FLAG-CMV (Sigma). The N-ter sequence was obtained by PCR using as template pcDNA3-Jamip1. The C-ter sequence, obtained by PCR using as template the yeast clone, retains the original HA tag. Both PCR products were subcloned in pcDNA4/V5-His/zeo (Invitrogen). To assay tyrosine phosphorylation (Fig. 5A), we used a C-ter expression plasmid in which the HA tag was removed by deleting a HindIII-BamHI fragment. GST-FERMJakI was obtained by subcloning the amplified human Jak1 FERM (aa 2-427) in pGEX-2T (Amersham Biosciences). Fragments derived from PCR were sequenced. Plasmid pcDNA4/V5-His-LacZ (Invitrogen) was used for of β-galactosidase expression. Northern Blot Analysis—Multiple tissue Northern membranes (Clontech) were probed with 3′ human (coding nucleotides 1228-1878, GenBank™ AK056126) and murine (coding nucleotides 1299-1878, GenBank™ AK077298) Jamip1 probes or a 3′ human Jamip2 probe (coding nucleotides 1784-2285, GenBank™ AB011127). An actin probe was used to assess equal loading. Membranes were hybridized as suggested by the manufacturer and exposed at -80 °C for 2-3 days for the Jamip probes and 3 h for actin. Cell Culture and Transfection—Human fibrosarcoma HT-1080 and 293T cells were kept in Dulbecco's modified Eagle's medium and 10% fetal calf serum. Jurkat cells were kept in RPMI 1640 medium and 10% fetal calf serum. Unless otherwise stated, transfections were performed by calcium phosphate precipitation (11Ragimbeau J. Dondi E. Alcover A. Eid P. Uze G. Pellegrini S. EMBO J. 2003; 22: 537-547Crossref PubMed Scopus (166) Google Scholar). Stable N-ter and C-ter clones were derived from HT-1080 and selected in zeocin (800 mg/ml). Jamip1-expressing clones were selected in neomycin (400 mg/ml). Calyculin A was purchased from Alexis Corp., and nocodazole and Taxol were from Sigma. Antibodies and Protein Analysis—Rabbit polyclonal antisera J1269-286 and J1609-626 were generated against Jamip1 peptides (aa 269-286 and aa 609-626). Antiserum J1269-286 was peptide affinity-purified. Anti-V5 mAb was from Invitrogen. mAbs specific for the FLAG and HA epitopes α-tubulin and acetylated tubulin were from Sigma, anti-β-tubulin was from Roche Applied Science, 4G10 was from UBI, the anti-Tyk2 T10-2 mAb was as described (11Ragimbeau J. Dondi E. Alcover A. Eid P. Uze G. Pellegrini S. EMBO J. 2003; 22: 537-547Crossref PubMed Scopus (166) Google Scholar), and the anti-Jak1 mAb (MAB3700) was from Chemicon International. Cell lysates were prepared as described (13Gauzzi M.C. Velazquez L. McKendry R. Mogensen K.E. Fellous M. Pellegrini S. J. Biol. Chem. 1996; 271: 20494-20500Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). For co-immunoprecipitation, cells were lysed in 0.5% Triton-X100, 20 mm Tris-HCl, pH 7.2, 250 mm NaCl, 5 mm EDTA, and protease inhibitors. Immunoblots were revealed with an ECL detection system (Amersham Biosciences). Bands were quantified by scanning with the Kodak Image Station 440CF. Luciferase Reporter Assay—293T cells (2 × 105 per 35-mm dish) were transfected with FuGENE 6 (Roche). The ISG54-luciferase construct (100 ng) was co-transfected with the pcDNA4 vector, Jamip1 (1 μg), N-ter (0.5 μg), or C-ter (2 μg). After 48 h, cells were treated with recombinant IFN-α2b (from D. Gewert). Luciferase activity was quantified in a luminometer (EG & E Berthold) as described (14Dondi E. Pattyn E. Lutfalla G. Van Ostade X. Uze G. Pellegrini S. Tavernier J. J. Biol. Chem. 2001; 276: 47004-47012Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In Vitro Interaction Assay—In vitro transcription-translation was performed using the TnT quick coupled transcription/translation system (Promega), according to the manufacturer's instructions, in the presence of l-[35S]methionine (1000 Ci/mmol) (Amersham Biosciences). In vitro translated proteins (2-10 μl) were incubated for 2 h at 4 °C with 20 μl of glutathione-Sepharose beads containing ∼3 μg of bound proteins in 100 μl of binding buffer (0.1% Nonidet P-40, 10% glycerol, 50 mm NaCl, 50 mm Tris-HCl, pH 8, and 1 mm dithiothreitol) with 0.5% bovine serum albumin and protease inhibitors. Beads were washed 3× in binding buffer and once in detergent-free buffer. Bound proteins were eluted in 20 μl of Laemmli buffer, resolved by SDS-PAGE, and visualized by autoradiography. Detergent Extraction Assay—Adherent cells were washed in PHEM (45 mm Pipes, pH 6.8, 45 mm Hepes, pH 6.8, 5 mm MgCl2, and 10 mm EGTA) and extracted for 5 min at room temperature with 400 μl of 0.5% Triton X-100 in PHEM and protease inhibitors. The supernatant (soluble) was collected. The detergent-insoluble matrix remaining on the dish was washed in PHEM, extracted in 400 μl of Laemmli buffer, scraped, and harvested (insoluble). For cells pretreated with drugs, all washes were with the drug. Equal volumes of soluble and insoluble samples were loaded on SDS-PAGE. Proteins were detected by Western blot. 5 × 106 Jurkat cells (5 × 105/ml) were treated with drugs, pelleted, transferred to 2-ml tubes, and extracted as described above, except that the soluble fraction was collected by centrifugation at 700 × g for 5 min. Fluorescent Microscopy—HT-1080 cells were transfected on glass coverslips with 1 μg of plasmid DNA and processed as described (15Ragimbeau J. Dondi E. Vasserot A. Romero P. Uze G. Pellegrini S. J. Biol. Chem. 2001; 276: 30812-30818Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Extraction prior to fixation was performed in 0.5% Triton X-100, 50 mm MES, 5 mm MgCl2, and 3 mm EGTA (16Kreis T.E. EMBO J. 1987; 6: 2597-2606Crossref PubMed Scopus (334) Google Scholar). Jurkat cells were seeded (4 × 105) on poly-l-lysine-coated coverslips, centrifuged for 1 min at 47 × g, fixed for 30 min at room temperature with 4% paraformaldehyde, and permeabilized with 0.05% saponin. Anti-V5 and anti-α-tubulin mAbs were used at 4 μg/ml, anti-β-tubulin mAb at 1 μg/ml, and anti-acetylated tubulin at 5 μg/ml. mAbs were revealed with Alexa594-coupled IgG1 secondary Abs (Molecular Probes) at 2 μg/ml. J1269-286 and J1609-626 antisera were used at 1:500 and 1:100 dilutions, respectively. Affinity purified J1269-286 Abs were used at 5 μg/ml. Rabbit primary Abs were revealed with Alexa488-coupled secondary Abs (Molecular Probes) at 4 μg/ml. Confocal microscopy analyses (Figs. 5A and 6, A and B) were performed with a Zeiss LSM-510 microscope, Z-series of optical sections were performed at 0.5 μm increments. Images were acquired with settings allowing the maximum signal detection below the saturation limits. A middle optical section is shown. For double staining, sequential acquisitions were performed to avoid fluorescence contamination between the two channels. For the other figures, visualization was with a Zeiss Axiovert 135 microscope (40× oil immersion lens). Images were captured with a Hamamatsu Orca II CCD camera and analyzed using AquaCosmos software. Cloning of Jamip1, a Member of a Novel Family—As a step further in the study of Jak proteins, we conducted a yeast two-hybrid screen to identify proteins interacting with the FERM homology domain of Tyk2. The bait comprised the entire FERM domain of Tyk2, and the cDNA library was from Jurkat T cells. One positive clone encoded a partial protein of 261 aa flanked by a 3′-untranslated region of 213 nucleotides. From data base searches, several matching human and murine expressed sequence tags were found, all originating from neural tissue. The entire coding sequence was cloned by reverse PCR using adult human brain cDNA as the template (see "Experimental Procedures"). The sequence encodes a protein of 626 aa with a predicted mass of 73.1 kDa that was designated Jamip1 (see below). Recently, Couve et al. reported the identification by yeast two-hybrid screen of a neuronal specific protein that can associate with the R1 subunit of the GABAB receptor (17Couve A. Restituito S. Brandon J.M. Charles K.J. Bawagan H. Freeman K.B. Pangalos M.N. Calver A.R. Moss S.J. J. Biol. Chem. 2004; 279: 13934-13943Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). This novel protein, named Marlin-1, is identical to Jamip1 (see "Discussion"). BLAST searches with the Jamip1 coding sequence predicted the existence of three highly related human genes with a conserved intron-exon organization (data not shown). Orthologues of the three Jamip genes were found in the murine genome. The chromosomal location and the available mRNA accession number of the human and the murine genes are shown in Table I. An alignment of the three predicted human proteins is shown in Fig. 1. Note that, at the present time, limited data base information is available for Jamip3, and its amino acid sequence was therefore derived by comparative analysis of the genomic sequence with the coding sequences of Jamip1 and Jamip2. Jamip1 shares 58% aa identity with Jamip2 and the predicted Jamip3. The aa identity between the predicted human and murine proteins ranges from 66% for Jamip3 to 97% for Jamip1. Three Jamip-related genes were found in fish, but none were found in Drosophila and Caenorhabditis elegans.Table IMembers of the Marlin/Jamip familyGene name/aliasChromosomal locationmRNA accession numberUnigeneHumanMouseHumanMouseHumanMouseMarlin1/Jamip14p16.25 B3 cMAK056126AK077298Hs. 101672Mm. 303004AY382340KIAA0555/Jamip25q3218 B3 cMAB011127XM_129010Hs. 43107Mm. 329404AF273057aPutative isoform.Jamip3bPredicted.10q26.112 A1.1 cMUnknownXM_137957UnknownUnknowna Putative isoform.b Predicted. Open table in a new tab Profile of Jamip1 and Jamip2 Expression—The mRNA expression profile of Jamip1 was analyzed in panels of human and murine tissues. Using 3′-specific Jamip1 probes, a single transcript of ∼2.5 kb was found highly expressed in adult brain and testis. Weaker signals were detected in other tissues (spleen, peripheral blood lymphocytes, lung, and intestine) (Fig. 2, A and B). Jamip2 expression in human tissues was also analyzed. A major transcript of >4 kb was highly expressed in brain, moderately expressed in thymus, spleen and lung, and weakly expressed in kidney, liver, and peripheral blood lymphocytes (Fig. 2A). Rabbit polyclonal antisera were raised against two Jamip1-specific peptides (Fig. 1). Immunoprecipitation/Western blot analyses using either antisera revealed a single 73-kDa band in Jurkat cells, T cell blasts, Daudi (B lymphoblasts), and NKL, a natural killer cell line. No protein was detected in HT-1080 fibrosarcoma, THP1 monocytic, or Madin-Darby canine kidney epithelial cells. The antisera revealed two specific bands in rat pheochromocytoma PC12 cells and mouse brain extract (Fig. 2D). Thus, Jamip1 appears predominantly expressed in lymphoid cells in addition to neural tissues. Jamip1 Can Homodimerize—Jamip proteins lack known conserved domains but are extremely rich in predicted α-helical coiled coils of varying lengths that are interrupted by non-helical regions ranging from 38 to 140 residues (Figs. 1 and 3A). Jamip1 contains two leucine zipper motifs, one of which is conserved in the family. Because coiled coil regions are known to serve as dimerization domains, we analyzed the ability of Jamip1, differentially tagged, to homodimerize in co-immunoprecipitation assays. FLAG-tagged Jamip1 was found to interact with V5-tagged Jamip1 but not with the control β-galactosidase protein (Fig. 3B, lanes 1 and 4). Truncated forms of Jamip1, comprising either the N-ter or the C-ter portion (Fig. 3A), were generated and tested in this assay. Both forms were found to associate with full-length Jamip1 (Fig. 3B, lanes 2 and 3). We also analyzed the ability of C-ter to interact with itself or with N-ter. As shown in Fig. 3C, the C-ter could self-associate but was unable to interact with the N-terminal region. Altogether, these data demonstrate that Jamip1 can homodimerize via the N-terminal and the C-terminal regions. Interaction between Jamip1 and Jak Proteins—To confirm the interaction between Jamip1 and Tyk2 in a context other than that of yeast, we performed co-immunoprecipitation of the two endogenous proteins from Jurkat T cells. As seen in Fig. 4A, endogenous Tyk2 (134 kDa) was brought down with the anti-Jamip1 serum. Moreover, in 293T cells endogenous Tyk2 was shown to associate with transfected Jamip1 and C-ter but not with N-ter (Fig. 4B). These results demonstrated that both endogenous and ectopically expressed Jamip1 could associate with endogenous Tyk2 via the C-terminal portion. Next, we asked whether Jamip1 could interact with the other three Jak proteins expressed in Jurkat cells. Experiments performed with a panel of rabbit anti-Jak2 and Jak3 antisera did not yield conclusive results. On the other hand, endogenous Jak1, detected with a monoclonal Ab, was shown to co-immunoprecipitate with Jamip1 (Fig. 4C). The ability of Jamip1 to interact specifically with the Jak1 FERM domain was also monitored. For this monitoring, the Jak1 FERM was expressed as a GST fusion protein and incubated with in vitro translated Jamip1 or control β-galactosidase. As shown in Fig. 4D, the fusion protein retained Jamip1 but not β-galactosidase. Thus, Jamip1 can interact with the FERM domains of both Tyk2 and Jak1. Given the well described involvement of Tyk2 and Jak1 in IFN-α/β signaling (18Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3388) Google Scholar), we investigated whether the forced expression of Jamip1 constructs in 293T cells interfered with IFN-α signaling. Cells were co-transfected with an IFN-inducible luciferase reporter and Jamip1, N-ter, or C-ter. At high IFN doses, a 50% decrease in luciferase induction was consistently observed in cells transfected with Jamip1 or C-ter (Fig. 4D). Thus, overexpression of Jamip1 perturbed the transcriptional response to high IFN doses, i.e. upon high receptor occupancy. This finding provided additional evidence of the capacity of ectopically expressed Jamip1 or C-ter to interact with endogenous Jak proteins. Jamip1 Associates with the Microtubule Cytoskeleton via the N-terminal Region—The subcellular localization of endogenous Jamip1 was studied in Jurkat T cells by confocal microscopy using affinity-purified anti-peptide polyclonal Abs. Staining was observed in organized filamentous structures that resembled the microtubule network as well as in non-fibrous structures enriched in the cortex areas. To monitor a possible association with microtubules, we co-stained Jamip1 and tubulin. A clear overlap was observed, with Jamip1 heavily decorating microtubules (Fig. 5A, sections a-c). Next, we analyzed HT-1080 cells transiently transfected with Jamip1. A mild Triton X-100 extraction was performed prior to fixation to remove cytosolic proteins and preserve membrane-cytoskeletal associations. Co-localization of Jamip1 with tubulin was evident using either anti-Jamip1 Abs (Fig. 5A, sections d-f) or anti-V5 mAb (data not shown). Both Jamip1 and tubulin filamentous staining patterns were disrupted by the addition of the microtubule-depolymerizing agent nocodazole (see Fig. 6B, sections a and b), demonstrating that Jamip1 and polymerized microtubules are closely associated. It was noteworthy that, whereas tubulin staining disappeared, Jamip1 collapsed in a diffuse, non-filamentous pattern. Thus, the solubilization property of the protein in Triton X-100 did not depend solely on the presence of assembled microtubules. To define which portion of Jamip1 directs its localization, we analyzed cells transfected with N-ter or C-ter. On cells permeabilized prior to fixation, N-ter was observed in association with stained interphase microtubules (Fig. 5B, sections a and b), and C-ter appeared predominantly nuclear (data not shown). In non-permeabilized cells the N-ter staining was unchanged, but C-ter appeared to be diffuse in the cytoplasm and the nucleus in a non-filamentous pattern (Fig. 5B, sections c and d). These results demonstrate that Jamip1 associates with the microtubule network via its N-terminal region. Jamip1 Influences Microtubule Dynamics—In the course of these studies we noticed that, in cells expressing a high level of N-ter, microtubules assumed a wavy appearance with a high density of closely spaced and looped bundles (Fig. 6A). To further analyze the effects of Jamip1 and N-ter on microtubule organization, transiently transfected cells were challenged with nocodazole. A gentle nocodazole treatment (1 μm for 10 min) was sufficient to induce a complete microtubule depolymerization in the majority of Jamip1-expressing cells, whereas filamentous structures, resulting from incomplete depolymerization, were visible in untransfected cells (Fig. 6B, sections a and b). This result was indicative of the enhanced nocodazole sensitivity of Jamip1-expressing cells. Conversely, in N-ter-expressing cells doubly stained circular microtubule bundles were preserved even upon a stronger nocodazole treatment (10 μm for 60 min) (Fig. 6B, sections c and d). To substantiate this result, we compared the nocodazole sensitivity of a stable N-ter clone and control cells. Lengthy residual tubulin polymers were consistently more abundant in N-ter-expressing cells than in control HT-1080 parental cells (Fig. 6C). Because modification of tubulin subunits by acetylation marks older and more stable microtubules (19Piperno G. LeDizet M. Chang X.J. J. Cell Biol. 1987; 104: 289-302Crossref PubMed Scopus (677) Google Scholar), we studied the effect of N-ter on microtubule stability by monitoring levels of acetylated tubulin. In N-ter-expressing cells, a strong acetylated tubulin staining was evident as compared with the weak or undetectable staining of untransfected cells (Fig. 6D). The interaction between Jamip1 and microtubules was further investigated by biochemical fractionation and analysis of the distribution of tubulin and Jamip1. We used a gentle extraction protocol that removes soluble proteins to leave intact the cytoskeleton framework and the associated proteins (20Infante C. Ramos-Morales F. Fedriani C. Bornens M. Rios R.M. J. Cell Biol. 1999; 145: 83-98Crossref PubMed Scopus (144) Google Scholar). In a stable clone, Jamip1 resided in the detergent-insoluble fraction, whereas tubulin was equally distributed between soluble and insoluble fractions (Fig. 7A, lanes 1 and 2). Treatment of cells with nocodazole (10 μm, 4 h) resulted in a nearly complete shift of tubulin from the insoluble to the soluble fraction, whereas only a minor pool of Jamip1 was solubilized (Fig. 7A, lanes 3 and 4). Endogenous Jamip1 behaved similarly, as assessed by the analysis of Jurkat cells (Fig. 7B). Thus, the distribution of Jamip1 only partly matched the distribution of tubulin, and its insolubility could only in part be attributed to its association with microtubules. Despite the drastic effect exerted by N-ter on microtubules (Fig. 6), when fractionation experiments were performed with cells transiently expressing N-ter, no changes in the distribution of tubulin could be observed with respect to control β-galactosidase transfected cells (Fig. 7C, upper sections). In view of the possibility that the fraction of N-ter-stabilized microtubules was too small to lead to a detectable shift in the distribution of total tubulin, we monitored the distribution of acetylated tubulin (Fig. 7C, lower sections). The amount of acetylated tubulin in the soluble fractions was comparable in the two transfected populations. However, the amount of acetylated, polymerized tubulin in the insoluble fractions was consistently higher in N-ter-transfected cells, and this finding was more evident upon nocodazole treatment (Fig. 7C, lower sections, compare lanes 3 and 4 with lanes 9 and 10). These results show that the N-terminal region is able to induce unique changes in microtubule organization, enhancing the pool of acetylated tubulin and reducing the sensitivity of microtubules to a depolymerizing agent. Both of these effects are indicative of an increased microtubule stability. It is well known that the activity of various microtubule-associated proteins and their affinity to microtub