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Interaction of p59fyn Kinase with the Dynein Light Chain, Tctex-1, and Colocalization During Cytokinesis This information is current as of June 18, 2017. Kerry S. Campbell, Suzanne Cooper, Mark Dessing, Sol Yates and Annie Buder J Immunol 1998; 161:1728-1737; ; http://www.jimmunol.org/content/161/4/1728 Subscription Permissions Email Alerts This article cites 65 articles, 39 of which you can access for free at: http://www.jimmunol.org/content/161/4/1728.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 References Interaction of p59fyn Kinase with the Dynein Light Chain, Tctex-1, and Colocalization During Cytokinesis1 Kerry S. Campbell,2 Suzanne Cooper, Mark Dessing, Sol Yates,3 and Annie Buder P rotein tyrosine kinase p59fyn (Fyn)4 is a 59-kDa member of the Src family of nonreceptor protein tyrosine kinases and is expressed predominantly in hemopoietic and neural tissues. Two distinct isoforms that are products of alternative splicing of separate seventh exons were originally defined by their selective expression in hemopoietic (FynT) and brain (FynB) cells (1). Fyn has been found to associate weakly with invariant chains of both TCR and B cell Ag receptor complexes in lymphocytes (2–5), and its enzymatic activity is increased upon cross-linking of either Ag receptor (6, 7). Fyn overexpression in T cell lines or thymocytes results in enhanced TCR signaling (8 –10), while FynT-deficient mice exhibit defects in TCR-stimulated signaling and proliferation of thymocytes, although more mature T cells exhibit improved responsiveness (11, 12). Although deficiency of the other major Src family member expressed in T lymphocytes, p56lck (Lck), more profoundly disrupts T cell development (13), mice that lack both FynT and Lck are entirely deficient in mature peripheral ab T lymphocytes (14, 15). Fyn kinase can efficiently couple both Ag receptors to intracellular calcium mobilization (11, 12, 16, 17), which may be mediated partially through direct tyrosine phosphorylation of the inositol 1,4,5-trisphosphate receptor (18). Fyn is also Basel Institute for Immunology, Basel, Switzerland Received for publication November 14, 1997. Accepted for publication April 15, 1998. 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. 1 The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche Ltd., Basel, Switzerland. 2 Address correspondence and reprint requests to Dr. Kerry S. Campbell, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111. E-mail address: [email protected] 3 Current address: Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. 4 Abbreviations used in this paper: Fyn, protein tyrosine kinase p59fyn; GST, glutathione S-transferase; Lck, protein tyrosine kinase p56lck; SH, Src homology; LynA, p56lyn; LynB, p53lyn; ITAMs, immunoreceptor tyrosine-based activation motifs; Blk, p55blk; pAb, polyclonal Ab; DB, DNA-binding (protein domain); TA, transcriptional activation (domain). Copyright © 1998 by The American Association of Immunologists involved in the function of IL-7 receptors (19, 20) and IL-5 receptors (21) in lymphocytes. Interestingly, Yasunaga et al. (22) have observed that the kinase also plays a critical role in mitotic division (cytokinesis) of proB cells. In addition, Ley et al. (23) have demonstrated significant cytoplasmic localization of Fyn in lymphocytes. These results strongly suggest that Fyn plays diverse cellular roles in addition to receptor-proximal signal transduction, and defining these alternative roles is an important challenge. Functional diversity between members of the Src family of kinases is mediated by four classical protein interaction domains. These Src homology (SH) domains are the kinase domain (SH1), which binds and phosphorylates substrate; SH2, which binds specific phosphotyrosine-containing sequences; SH3, which selectively binds proline rich sequences; and an amino (N)-terminal domain of 60 to 90 amino acids. Although the majority of Nterminal amino acids are unique for each kinase, the first 10-residue segment contains common elements and has been termed SH4 (24). This SH4 domain facilitates membrane localization through myristoylation of a glycine and palmitoylation of cysteines on most Src family members (24 –29). In addition, the SH4 domains of Fyn and p56lyn (LynA) have been reported to mediate weak interactions with unphosphorylated immunoreceptor tyrosinebased activation motifs (ITAMs) of lymphocyte Ag receptors (3, 30 –32) and the signaling effectors phospholipase C-g, GAP (GTPase-activating protein), and MAP (microtubule-associated protein) kinase (33), indicating that this domain also facilitates important protein interactions. Tctex-1 is the product of a gene within the t complex on chromosome 17 of mice, which was originally cloned from sperm cDNA as a potentially important distorter component of certain mutant t haplotypes (34). t haplotypes contain multiple conserved mutated genetic elements within the t complex that are responsible for various phenotypes of tail length, embryonic lethality, male sterility, and sperm cell transmission ratio distortion (35). Analysis of the Tctex-1 protein has been limited (36), until very recently, when King et al. (37) demonstrated that it is a light chain component of the cytoplasmic dynein complex. Cytoplasmic dynein is a large microtubule-based multicomponent ATP-dependent motor 0022-1767/98/$02.00 Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 The protein tyrosine kinase p59fyn (Fyn) plays important roles in both lymphocyte Ag receptor signaling and cytokinesis of proB cells. We utilized yeast two-hybrid cloning to identify the product of the tctex-1 gene as a protein that specifically interacts with Fyn, but not with other Src family kinases. Tctex-1 was recently identified as a component of the dynein cytoskeletal motor complex. The capacity of a Tctex-1-glutathione S-transferase fusion protein to effectively bind Fyn from cell lysates confirmed the authenticity of this interaction. Tctex-1 binding required the first 19 amino acids of Fyn and integrity of two lysine residues within this sequence that were previously shown to be important for Fyn interactions with the immunoreceptor tyrosine-based activation motifs (ITAMs) of lymphocyte Ag receptors. Expression of tctex-1 mRNA and protein was observed in all lymphoma lines analyzed, and immunofluorescence confocal microscopy localized the protein to the perinuclear region. Analysis of a T cell hybridoma revealed prominent colocalization of Tctex-1 and Fyn at the cleavage furrow and mitotic spindles in cells undergoing cytokinesis. Our results provide a unique insight into a mechanism by which Tctex-1 might mediate specific recruitment of Fyn to the dynein complex in lymphocytes, which may be a critical event in mediating the previously defined role of Fyn in cytokinesis. The Journal of Immunology, 1998, 161: 1728 –1737. The Journal of Immunology Materials and Methods Mammalian cell lines Cell lines and their sources were the murine proB cell line 38B9 (Dr. A. Rolink, Basel, Switzerland); cytotoxic T cell clone CTLL-2 (Dr. J. Garcı́aSanz, Madrid, Spain); COS-7 cells (Dr. B. Imhof, Geneva, Switzerland); the murine B cell lymphomas K46, A20, and WEHI-231.7; and NIH 3T3 cells transfected with the ZIP-Fyn plasmid to express high levels of p59fyn protein (45) (all from Dr. J. Cambier, Denver, CO). The fyn-transfected murine T cell hybridoma N17 (10) was generously provided by Dr. T. Yamamoto and colleagues (University of Tokyo, Japan). Cells were cultured as previously described (46). Geneticin (G-418; Life Technologies, Gaithersburg, MD) was supplemented in cultures of COS-7 transfectants (500 mg/ml) and N17 cells (770 mg/ml). Antibodies Anti-Tctex-1 polyclonal Abs (pAb) were generated in rabbits against purified full length murine Tctex-1, which was produced in Escherichia coli as a GST fusion protein (see below) and cleaved from the GST with human thrombin (Sigma). The Tctex-1-specific Ab was affinity purified using the cleaved protein on CNBr-activated Sepharose (Pharmacia, Uppsala, Sweden), eluted with 3.5 M MgCl2 (pH 7.2), dialyzed against PBS, and stored at 270°C. Anti-Fyn Abs (both from Santa Cruz Biotechnology, Santa Cruz, CA) were a mAb (sc-434, mouse IgG1; agarose conjugate used for immunoprecipitation or unconjugated for intracellular staining) and a rabbit pAb (sc-016, unconjugated, used for immunoblotting and intracellular staining). Anti-FLAG mAb (M2; mouse IgG1; Kodak, Rochester, NY) was conjugated to CNBr-activated Sepharose for immunoprecipitation. Antib-tubulin mAb was from Boehringer Mannheim (Mannheim, Germany; mouse IgG2b). Anti-dynein 74-kDa intermediate chain Ab, clone 74.1 (47) (mouse IgG2b), was purchased from Chemicon (Temecula, CA). The antihuman MHC class I mAb, W6/32, was used as a hybridoma culture supernatant and was generously provided by Dr. Marina Cella (Basel Institute for Immunology). Rabbit anti-mouse IgG pAb, goat anti-rabbit IgG conjugated with Texas Red or FITC, and goat anti-mouse IgG1 conjugated with Texas Red were from Southern Biotechnology Associates (Birmingham, AL). Oregon Green 488-conjugated goat anti-rabbit IgG Ab was from Molecular Probes (Eugene, OR). Yeast two-hybrid method A Gal4-based yeast two-hybrid system was generously made available by Drs. P. Chevray and D. Nathans (Johns Hopkins University, Baltimore, MD) (48). The pPC62 plasmid encodes the DNA-binding domain of Gal4 (amino acids 1–147) with 39 SalI and NotI restriction sites, which were utilized to generate “bait” fusion protein constructs. Bait constructs were generated by PCR from a K46 murine B cell lymphoma cDNA library (described below); these were the N-terminal domains of Fyn (amino acids 1– 85, a mutated version with lysines 7 and 9 mutated to alanines, and truncated versions with indicated amino acids), p55blk (Blk; amino acids 1–56), LynA (amino acids 1– 68), p53lyn (lynB; amino acids 1– 47), the cytoplasmic domain of Ig-b (amino acids 186 –228), and full length Tctex-1 (amino acids 1–113). The pPC86 plasmid encodes the Gal4 transcriptional activation domain (amino acids 767– 881) and 39 restriction sites for generation of “prey” fusion protein constructs. A cDNA library was prepared from 5 mg of poly(A)1-enriched RNA that had been isolated from the K46 B cell lymphoma using the Poly(A) Quik Kit (Stratagene, La Jolla, CA). One half of the K46 cells were resting, while the other half had been stimulated with sheep anti-mouse IgG (Silenus, Hawthorne, Australia; 1 mg/ml) and harvested at time points between 15 min and 6 h. Directionally cloned cDNA was generated with the ZAP-cDNA synthesis kit (Stratagene) and inserted into EcoRI/SalI-modified pPC86 plasmid (provided by Dr. T. Watanabe, Kyushu University, Fukuoka, Japan). The library contained about 1.2 million original clones with .95% inserts ranging from 200 to 2000 base pair. Additional prey fusion protein constructs that were generated by PCR into pPC86 were Tctex-1 truncations (amino acid sequences described in the text), the cytoplasmic domain of Ig-a (amino acids 160 –220), and the N-terminal domains of Fyn (amino acids 1– 85), LynA (amino acids 1– 68), and Lck (amino acids 1– 67). The yeast reporter strain was HF7c (generously provided by Dr. D. Beach, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) with integrated growth selection (HIS3) and b-galactosidase (lacZ) reporter genes (49). For cDNA library screening, yeast were first transfected with the bait plasmid (in pPC62) and subsequently transfected with a library (in pPC86) using lithium acetate and polyethylene glycol (50). Colonies that grew in the absence of histidine were secondarily screened for b-galactosidase activity (51). cDNA plasmids were released from positive yeast colonies and cloned as described (52) and sequenced by PCR using the SequiTherm cycle sequencing kit (Epicentre Technologies, Madison, WI). Transfectants were streaked on agarose medium lacking histidine to test for growth reporter activation or on medium containing histidine to test for activation of the lacZ reporter. Northern blotting Total RNA (10 mg/sample) was isolated with guanidinium thiocyanate from cell lines or mouse tissues (C57BL/6 mice from Iffa Credo, L’Arbresle, France), separated on a 1% agarose gel, and transferred to a nylon membrane (Hybond-N; Amersham, Arlington Heights, IL). A full length tctex-1 cDNA sequence probe and a full length b-actin sequence probe were generated by PCR and labeled with [32P]dCTP using the Prime-It random primer kit (Stratagene). The membrane blot was hybridized at 65°C and washed at 65°C. Immunoblotting Immunoprecipitates or whole cell lysates (lysed directly in Laemmli buffer) were separated on SDS-PAGE and electrophoretically transferred to Immobilon-P membranes (Millipore, Bedford, MA). Immunoblotting and stripping were performed as previously described (53). Abs were rabbit anti-Tctex-1 (10 mg/ml) or rabbit anti-Fyn pAb (1 mg/ml). Secondary reagent was 125I-labeled protein A (0.5 mCi/ml; Amersham), and proteins were visualized by autoradiography. It should be noted that detection of Tctex-1 can be obscured by a background band at 14 kDa in lymphocyte whole cell lysates when probed with horseradish peroxidase-conjugated secondary reagents; this result can be avoided by using 125I-labeled protein A. Metabolic labeling, protein precipitations, and in vitro kinase reactions COS-7 cells were metabolically pulse-labeled in some studies for 15 to 45 min with 5 mCi of [35S]cysteine/methionine (Amersham) in 20 ml of cysteine/methionine-free Dulbecco’s modified Eagle’s medium and chased for 2 to 9 h in normal medium. Cells were lysed for 30 min on ice in 1% digitonin buffer, 1% Triton X-100 (Surfact-Amps; Pierce, Rockford, IL) buffer, or RIPA buffer (1% Triton X-100, 0.1% sodium deoxycholate (Merck, Darmstadt, Germany)), and 0.1% SDS (Bio-Rad, Hercules, CA), each containing 75 mM NaCl, 10 mM Tris (pH 7.4), 10 mM NaF, 0.4 mM EDTA, 1 mM Pefabloc SC (Boehringer Mannheim), 2 mM sodium orthovanadate, and 1 mg/ml each of aprotinin, soybean trypsin inhibitor, and leupeptin (reagents were from Sigma unless otherwise noted). Lysates were microfuged at 14,000 rpm for 15 min and subjected to precipitation for 2 to 4 h with GST fusion proteins (10 mg/sample) or Abs (2–5 mg/sample) Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 unit that plays important roles in intracellular retrograde organelle transport, membrane trafficking, mitotic spindle localization, and centrosome separation during mitosis (38 – 40). Tctex-1 has also recently been shown to be a component in inner arm I1 of flagellar dynein (41). A human homologue of the tctex-1 gene was reported to encode 94% protein homology to the murine protein (42), and a related human gene, “candidate RP3,” encodes a protein with 55% amino acid identity to human Tctex-1 (43). A homologue of another mouse t complex-encoded protein, Tctex-2, which has limited sequence similarity to Tctex-1, was also recently identified as a light chain component of flagellar dynein in Chlamydomonas (44), and a Tctex-1 homologue in the same species exhibits 60% identity to the mouse protein (41). To better define functionally important protein interactions with the unique N-terminal domain of Fyn, we have utilized the yeast two-hybrid technique. Using this system, we could not demonstrate direct Fyn interactions with the ITAM-containing B cell Ag receptor components, Ig-a or Ig-b. Upon screening a B cell cDNA library, however, Tctex-1 was recognized as a strong Fyn-binding protein in the yeast system, and this interaction was localized to the first 19 amino acids of Fyn. The validity of this protein-protein interaction was confirmed by the capacity of a Tctex-1 fusion protein to bind Fyn from cell lysates. Fyn and Tctex-1 were found to colocalize during cytokinesis in a T cell hybridoma, thereby suggesting that the interaction with Tctex-1 can selectively recruit Fyn to the dynein motor complex during mitosis of lymphocytes. 1729 1730 Fyn INTERACTS WITH THE DYNEIN LIGHT CHAIN, Tctex-1 FIGURE 1. Tctex-1 can interact specifically with the N-terminal domain of Fyn. Yeast were transfected with various combinations of plasmid constructs encoding the indicated Gal4 DB fusion proteins and Gal4 TA fusion proteins. Transfectants were subsequently streaked on agarose media containing (1) or lacking (2) histidine. Yeast transfectants that grow without histidine demonstrate an interaction between the fusion protein partners that recruits the TA to the reporter gene and drives its transcription to permit growth. Fusion protein partners were Tctex-1, the N-terminal domains (-NH) of Fyn, Blk, LynA, and LynB, and the cytoplasmic domains (cyto.) of Ig-a and Ig-b. GST and epitope-tagged fusion proteins and protein expression GST fusion protein constructs of the Fyn N-terminal domain (amino acids 1– 85) and Tctex-1 (amino acids 1–113) were generated by excision of these cDNAs from pPC62 and pPC86 and ligation into pGEX-4T-2 plasmid (Pharmacia). Other GST fusion constructs were human platelet-derived growth factor receptor kinase insert region (amino acids 698 –797 in pGEX-3X; provided by Dr. A. Kazlauskas, Harvard University) and cytoplasmic domains of murine Ig-a and Ig-b (3) (provided by Dr. J. Cambier). E. coli (Top10F9) were transformed with these plasmids and induced for 2 to 4 h with 0.3 mM isopropyl b-D-thiogalactopyranoside as described (3). Cells were pelleted, and probe sonicated on ice in 10 ml of Tris-buffered saline (TBS; 10 mM Tris, pH 7.4, 150 mM NaCl) containing Complete protease inhibitor (Boehringer Mannheim; 1 tablet/50 ml TBS), admixed to 1% Triton X-100 and pelleted at 12,000 rpm for 15 min. Cleared lysate was precipitated for 2 h to overnight with glutathione-Sepharose 4B beads (Pharmacia), and beads were washed with 1% Triton X-100 in TBS and stored at 4°C as a 50% slurry in the same buffer. A C-terminal FLAG epitope-tagged Tctex-1 construct was created by PCR encoding DYKD DDDK-stop after the 113-amino acid murine Tctex-1 sequence and cloned into the mammalian expression plasmid, pcDNA3 (InVitrogen, San Diego, CA). COS-7 cells (70% confluent) were transfected in 100-mm petri dishes with 5 mg of plasmid DNA using Dosper liposomal transfection reagent according to the manufacturer’s protocol (Boehringer Mannheim), and protein expression was assayed two days later. Stably integrated clones were isolated by limiting dilution. An expression plasmid encoding N-terminal FLAG-tagged Tctex-1 was also generated (M-DYKDDDDK-), but did not produce significant levels of protein expression in COS-7. Immunofluorescence confocal microscopy Some cells were grown on chamber slides (see Fig. 7A) or allowed to adhere on poly-L-lysine coated slides (Fig. 7B) before staining. Other cells were stained in suspension after sorting and subsequently cytospun onto slides (Fig. 7C). The following staining protocol was utilized, since it resulted in optimal retention of b-tubulin structure in cells. Cells were washed in 37°C serum-free Iscove’s modified Dulbecco’s medium before fixing at 37°C for 10 min with 3% paraformaldehyde (in PBS, pH 7.0, and 5.4% glucose). Subsequent steps were performed at room temperature. After washing twice in PBS (pH 7.4), fixed cells were permeabilized for 30 min in PBS/saponin (PBS with 0.1% saponin and 1 mM HEPES) and washed again in the same. Cells were blocked for 30 min in PBS/saponin/BSA (PBS/saponin with 2% BSA) and washed once in PBS/saponin. Cells were then incubated for 30 to 45 min with optimal concentrations of primary Ab in PBS/saponin/BSA and washed three times with PBS/saponin. An optimal concentration of secondary Ab in PBS/saponin/BSA was added for 30 to 45 min, and cells were washed three times with PBS/saponin, followed by three washes with PBS. Some cells were fixed, stained with Hoechst 33342 (Molecular Probes), and sorted on a FACS Vantage into G0/G1, S, and G2/M stages (55) before intracellular Ab staining (Fig. 7C). Coverslips were mounted with Mowiol (Hoechst, Frankfurt, Germany) or Fluoromount-G (Southern Biotechnology) mounting solution and analyzed through a 633/1.4 Zeiss Plan-Apochromat lens on a Zeiss Axiovert 100 inverted microscope fitted with a Bio-Rad MRC 1024 laser scanning confocal imaging system. Images were acquired in accumulation mode, analyzed using LaserSharp software (Bio-Rad, version 2.1A), and processed with Adobe Photoshop (version 3.0, Adobe Systems, San Jose, CA). Compensation was rigidly controlled using single-stained cell samples and/or independent sequential excitation of each fluorescent dye to assure lack of bleedover between the green channel and the red channel. Pretreatment of the anti-Tctex-1 pAb preparation with recombinant Tctex-1 protein completely eliminated immunofluorescent reactivity, thereby indicating specificity of the staining. Results Identification of Tctex-1 as a Fyn-binding protein Previous experiments with GST fusion proteins have suggested that the N-terminal domains of Fyn and LynA can interact directly with the cytoplasmic domains of the B cell Ag receptor chains, Ig-a and Ig-b (31). As demonstrated in Figure 1, we could not detect any direct protein interactions between these kinase N-terminal domains (or LynB; data not shown) and either Ig-a or Ig-b cytoplasmic domains in a Gal4-based yeast two-hybrid system in either orientation (DB or TA fusions), nor in a more sensitive LexA-based system (data not shown; sensitivity described in Ref. 46). All fusion proteins of appropriate size were produced in the yeast transfectants, however, as assessed by anti-GAL4 immunoblotting (data not shown). Although these results suggest that Fyn cannot directly interact with Ig-a or Ig-b, one must consider that many factors can contribute to a negative result in this assay, and Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 prebound to glutathione-Sepharose, GammaBindPlus Sepharose, or protein A-Sepharose 4 Fast Flow beads (Pharmacia). Protein precipitations were washed five times with 0.2% digitonin or RIPA buffer (as above). Some samples were subjected to in vitro kinase reactions with [32P]ATP, quenched with 1% Triton X-100 buffer (as above), and secondarily immunoprecipitated with anti-Fyn mAb-coupled agarose as previously described (46). Phosphoamino acid analysis was performed as previously described (54). The Journal of Immunology therefore, this is not proof that these interactions cannot occur in vivo. To identify additional candidate proteins that interact efficiently with the Fyn N-terminal domain, we screened a cDNA library from the K46 B cell lymphoma using this domain as bait in the two-hybrid system. The majority of yeast colonies that scored positive in this screen contained the full length tctex-1 cDNA (34), which encodes a protein component of the ATP-dependent dynein motor complex (37). The interaction with Fyn was specific, as demonstrated in Figure 1, since the tctex-1 gene product did not interact with the N-terminal domains of LynA, LynB, Blk, or p56lck (Lck; see Fig. 5) as measured by activation of either His3 (histidine-free growth selection) or lacZ (b-galactosidase; data not shown) reporters. In addition, the N-terminal domain of Fyn was not observed to form homotypic interactions (Fig. 1). Tctex-1 failed to interact with Ig-a or Ig-b cytoplasmic domains in this assay (data not shown), and we could not demonstrate any capacity of Tctex-1 to “couple” Ig-a to Fyn when expressed as a third protein in yeast two-hybrid experiments (data not shown). The interaction of Fyn with Tctex-1 is clearly a strong protein-protein interaction when compared with the lack of detectable interactions between Fyn and Ig-a or Ig-b. lacking. As shown in Figure 2, Northern blot analysis demonstrated ample expression of tctex-1 mRNA in all tissues examined from BDF1 mice. Thymic expression was more pronounced than that of spleen, while significant expression of tctex-1 mRNA was observed in both B and T lymphocyte lines (Fig. 2). Less abundant expression of tctex-1 was also evident in the brain, suggesting that the protein is also available for interaction with Fyn in neuronal cells (Fig. 2). An affinity-purified pAb against recombinant Tctex-1 was prepared to test lymphocyte cell lines for protein expression. To characterize this pAb preparation, a C-terminal FLAG epitope-tagged version of Tctex-1 was stably expressed in COS-7 cells. As shown in Figure 3a, immunoprecipitation from a 35S metabolically labeled transfectant (clone C20) with either anti-Tctex-1 or antiFLAG Abs resulted in the purification of a specific band of about 15 kDa. This apparent mass is slightly higher than that predicted for Tctex-1 (;14 kDa) due to the epitope tag. This band was the major specific protein immunoprecipitated by the anti-Tctex-1 pAb preparation when compared with control immunoprecipitations (anti-human MHC class I and anti-tubulin mAbs in lanes 3 and 4 of Fig. 3a). In addition, the pAb preparation and anti-FLAG mAb reproducibly immunoprecipitated similar amounts of Tctex-1. Several murine B and T cell lines were tested for Tctex-1 expression using the pAb preparation. Although Tctex-1 was difficult to detect by immunoblotting with the pAb in whole cell lysates of lymphocyte lines (one to two million cell equivalents per lane; data not shown), it could be readily immunoprecipitated with this Ab preparation from several lymphocyte cell lines and detected by immunoblotting with the same Ab. The protein was observed in immunoprecipitates from 50 to 70 million cell equivalents of the fyn-transfected T cell hybridoma, N17 (10), and the B cell lymphomas K46, A20, and WEHI 231.7 (Fig. 3b; data not shown). The specific reactive band was observed at 14 kDa, as predicted, and this migration corresponded exactly to that of recombinant Tctex-1 as shown in Figure 3b. No significant improvement in the amount of Tctex-1 immunoprecipited from lysates of K46 cells could be demonstrated upon lysis with the addition of other nonionic detergents, harsher RIPA buffer, 10 mM ATP, or nocodazole pretreatment of cells, and only slight improvement was achieved using a monoclonal anti-dynein intermediate chain Ab (data not shown). Therefore, both mRNA and the protein product of the tctex-1 gene were detectably expressed in hemopoietic tissues as well as T and B lymphocyte lines, but protein levels were routinely low in these cells. Despite significant efforts, we could not detectably coimmunoprecipitate the two proteins from Fyn-transfectants of COS-7 cells, NIH 3T3 fibroblasts, or the T cell hybridoma N17 by immunoprecipitation of either protein or dynein intermediate chain (data not shown). Detergents tested in these studies included Triton X-100, digitonin, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate), and RIPA, and detection methods employed were either 35S metabolic labeling, immunoblotting, or in vitro phosphorylation of immunoprecipitated kinase in the presence of [32P]ATP. These results suggest that the protein interaction is either transient in these cells or the off rate of the interaction is high, thereby limiting detection after washing of immunoprecipitates. Expression of Tctex-1 in lymphoid cells The tctex-1 gene had been cloned from sperm cDNA and identified as a product of the t complex of mice (34). Previous analysis by Northern blotting had determined that the gene was strongly expressed in the testes and ovaries (34) and weakly in the thymus of wild-type mice, but a detailed analysis in hemopoietic tissues was Fyn interaction with Tctex-1-GST fusion protein in vitro Due to the difficulties in demonstrating coimmunoprecipitation, we tested the capacity of recombinant Tctex-1-GST fusion protein to interact with Fyn in cell lysates. Digitonin lysates of Fyn-transfected NIH 3T3 cells (45) were adsorbed with various GST fusion Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 2. Tctex-1 mRNA is expressed in all tissues and immune cell lines tested. Northern blot of tctex-1 expression in various tissues and lymphocyte lines. Total RNA (10 mg/lane) was sequentially hybridized with the full length tctex-1 cDNA (top) and b-actin cDNA (bottom). The various murine cell lines tested are representative of proB cells (38B9), immature B cells (WEHI-231.7), mature B cells (K46), and mature T cells (CTLL-2). 1731 1732 Fyn INTERACTS WITH THE DYNEIN LIGHT CHAIN, Tctex-1 proteins, including Tctex-1, and precipitates were washed and subjected to in vitro [32P]ATP phosphorylation reactions to detect associated phosphoproteins. As presented in Figure 4a, GST fusion proteins of the cytoplasmic domains of Ig-a, and to a lesser extent, Ig-b, were significantly phosphorylated in this assay, while Tctex-1-GST fusion protein was only weakly phosphorylated. The Tctex-1-GST fusion protein, however, selectively coprecipitated a phosphoprotein band of about 59 kDa, which comigrated with p59fyn. Reimmunoprecipitation from these phosphorylation reactions with an anti-Fyn mAb confirmed that the kinase had associated with the Tctex-1-GST fusion protein (Fig. 4a). The [32P]p59 band from Figure 4a exclusively contained phosphotyrosine, which further verified its identity as autophosphorylated Fyn (Fig. 4b). Although Tctex-1-GST did not provide a good substrate for the adsorbed Fyn in this assay, the minimal 32P labeling of Tctex1-GST was also found to be exclusively incorporated onto tyrosine residues (Fig. 4b). These results suggest that Tctex-1 can be weakly tyrosine phosphorylated by Fyn kinase. Finally, Fyn binding to Tctex-1-GST was confirmed by immunoblotting with anti-Fyn Ab as demonstrated in Figure 4c. The Ig-a-GST fusion protein was also able to adsorb Fyn in both of these assays as has previously been reported (3), while Ig-b, PDGFR-KI, and the Nterminal domain of Fyn did not bind appreciable amounts of the enzyme. In summary, the Tctex-1-GST fusion protein can selectively bind Fyn from digitonin lysates in vitro, thereby confirming the yeast two-hybrid interaction. Truncation analysis defining interacting sequences in Fyn and Tctex-1 To identify the specific interacting domains in both Fyn and Tctex-1, truncation analysis studies were undertaken in the yeast two-hybrid system. When truncations of the 85-amino acid N-terminal domain of Fyn were tested as demonstrated in Figure 5a, elimination of just the first 6 amino acids from the N terminus completely abrogated the interaction. The C-terminal amino acids of this domain, however, did not appear to contribute to the interaction with Tctex-1, yet amino acids 1 through 19 still exhibited strong interaction. On the other hand, further truncation to amino acids 1 through 10 completely abolished the interaction (see Fig. 5a). Previous studies by Timson Gauen et al. (32) have indicated that amino acids 1 through 10, and in particular the lysine residues at positions 7 and 9 of Fyn (Fig. 5b), are critical elements in the interaction of this domain with ITAM-containing sequences on lymphocyte Ag receptors. We also tested for involvement of lysines 7 and 9 in interaction with Tctex-1 by mutating them to alanines in the complete N-terminal domain. As shown in Figure 5c, this mutated fragment of Fyn did not interact with Tctex-1, indicating that these lysine residues are involved in interactions of the kinase with both ITAM-containing sequences and Tctex-1. Thus, Tctex-1 interacts with the first 19 amino acids of Fyn; lysine residues at positions 7 and 9 are critical elements in this interaction domain, although they are clearly not the only binding residues, as determined in the truncation analysis (Fig. 5a). Tctex-1 was also truncated and tested for interaction with Fyn in the two-hybrid system. As shown in Figure 6, Tctex-1 was very sensitive to truncation from both ends, since truncation of amino acids 105 through 113 completely abolished the interaction with Fyn, and elimination of amino acids 1 through 12 significantly reduced the interaction. These results demonstrate that the protein requires integrity of both termini to form the Fyn-interacting structural domain. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 3. Analysis of Tctex-1 protein expression in lymphocytes. a, Affinity-purified anti-Tctex-1 pAb specifically immunoprecipitates FLAG epitopetagged Tctex-1. COS-7 cells were stably transfected with a C-terminal FLAG-tagged Tctex-1 expression plasmid (clone C20), metabolically labeled with [35S]cysteine/methionine, lysed in 1% digitonin, and immunoprecipitated with anti-FLAG mAb covalently conjugated to Sepharose beads (15 mg/30 ml of beads) or GammaBindPlus Sepharose beads (30 ml) precoupled with anti-Tctex-1 pAb (3 mg), anti-human MHC class I mAb (W6/32, labeled huMHC-I; 75 ml of culture supernatant), or anti-tubulin mAb (3 mg). Immunoprecipitated proteins were separated on 17% SDS-PAGE, transferred to Immobilon-P membrane, and detected by autoradiography. b, Immunoprecipitation of Tctex-1 from lymphocyte lines. The K46 (55 million/sample) and A20 B cell lymphomas and the N17 T cell hybridoma (70 million each per sample) were lysed in 1% digitonin and immunoprecipitated with rabbit anti-mouse IgG pAb, affinity-purified anti-Tctex-1 pAb (each 10 mg/sample), or preimmune serum from the same rabbit (10 ml), which were precoupled to 30 ml of GammaBindPlus Sepharose beads. Immunoprecipitates were separated on 17% reducing SDS-PAGE, transferred to Immobilon-P membranes, and immunoblotted with anti-Tctex-1 pAb (10 mg/ml) and 125I-labeled protein A. The 16-kDa m.w. marker (lysozyme) routinely cross-reacted with anti-Tctex-1 pAb as evident in the “marker” lane. The Journal of Immunology FIGURE 5. Truncation mapping of the domain within the N terminus of Fyn that interacts with Tctex-1. a, The first 19 amino acids of Fyn interact with Tctex-1. Truncation mutants of the N terminus of Fyn were tested as Gal4 DB domain fusions for interaction with full length Tctex-1 fused to Gal4 TA domain. Tctex-1 fused to the Gal4 DB domain was also tested for interaction with the N-terminal domain of Lck kinase. Transfectants were tested for activation of a histidine-free growth reporter and a b-galactosidase reporter. Magnitudes of reporter activation are indicated (11 5 strong, 2 5 none detected). b, Sequence of amino acids 1 through 19 of murine Fyn that interacts with Tctex-1. Lysines 7 and 9, myristoylated glycine 2 and palmitoylated cysteines 3 and 6 are marked. c, Mutation of lysines 7 and 9 of the N terminus (-NH) of Fyn abrogates interaction with Tctex-1. Mutant Fyn N-terminal domain (amino acids 1– 85) with lysines 7 and 9 changed to alanines (KK/AA) was compared with wild-type Fyn domain (amino acids 1– 85) for interaction with Tctex-1 or the N-terminal domain of Lck in the histidine-free growth assay. Intracellular colocalization of Tctex-1 and Fyn proteins in T lymphocytes during cytokinesis Confocal immunofluorescence analysis was performed to determine the intracellular localization of Tctex-1. Analysis of C-FLAG-Tctex-1-transfected COS-7 cells demonstrated that antiFLAG mAb (Fig. 7A) and anti-Tctex-1 pAb (identical pattern; data not shown) diffusely stained the cytoplasm with the majority of staining concentrated in the perinuclear region. Alternatively, microtubules (stained with anti-b-tubulin) emanated from the perinuclear Tctex-1 stained region and extended to the periphery of the Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 4. Fyn kinase binds to a GST-fusion protein of Tctex-1 in vitro. a, Fyn selectively binds to Tctex-1-GST fusion protein in vitro. Digitonin lysates of fyn-transfected fibroblasts (ZIP-Fyn cells; 2.5 3 106/sample) were adsorbed with the indicated GST fusion proteins or anti-Fyn mAb and subjected to in vitro kinase reactions. Ten percent was retained to analyze primary precipitations (left panel), and the remainder was reimmunoprecipitated with anti-Fyn mAb (right panel). Fusion proteins were GST fused to cytoplasmic domains of Ig-a or Ig-b, kinase insert region of platelet-derived growth factor receptor (PDGFR KI), full length Tctex-1, or amino acids 1 through 85 of Fyn (Fyn-NH). Phosphoprotein bands corresponding to the Tctex-1-GST fusion protein (open arrow) and the p59fyn band reimmunoprecipitated from the Tctex-1-GST precipitation (closed arrow) are marked. b, GST-Tctex-1 and associated p59fyn are exclusively phosphorylated on tyrosine residues. The marked 32P-labeled phosphoprotein bands in panel a were subjected to phosphoamino acid analysis. c, Anti-Fyn imunoblotting confirmed Fyn binding to Tctex-1-GST. Digitonin lysates of ZIP-Fyn cells (14 3 106/sample) were adsorbed with GST fusion proteins or anti-Fyn mAb. SDS-PAGE-separated samples were immunoblotted with anti-Fyn pAb. 1733 1734 Fyn INTERACTS WITH THE DYNEIN LIGHT CHAIN, Tctex-1 Tctex-1 at the center of the cleavage furrow in the cell crosssections as presented in Figure 7C. Overall, this stage of cell division was the only interval during which we consistently observed intracellular colocalization of Fyn with Tctex-1. Tctex-1 staining was always perinuclear throughout all other stages of the cell cycle, and we never observed staining of the nucleus or plasma membrane. In conclusion, we observed highly reproducible colocalization of Fyn and Tctex-1 during cytokinesis at both the cleavage furrow and mitotic spindles in this T cell hybridoma. Discussion cells (Fig. 7A). Tctex-1 staining was not detected in the nucleus or plasma membrane. Double-staining intracellular immunofluorescence studies were also performed to determine whether Tctex-1 and Fyn are colocalized in lymphocytes. The fyn-transfected murine T cell hybridoma, N17 (10), provided detectable levels of kinase for these studies. Elevated Fyn expression in N17 cells was clearly evident in immunoblots of whole cell lysates and immunofluorescent staining when compared with other lymphocyte cell lines (data not shown). Most N17 cells exhibited Fyn staining that was predominantly concentrated to the plasma membrane, although some cells exhibited significant foci of Fyn staining within the cytoplasm (Fig. 7B) as previously reported by Ley et al. (23). Although Tctex-1 staining was concentrated within the perinuclear/cytoplasmic region and generally distinct from Fyn-stained regions, a subset (;25%) of the T hybridoma cells demonstrated distinct cytoplasmic foci of Fyn, some of which were clearly colocalized with Tctex-1 (Fig. 7B). We surmised that this inconsistent colocalization of Fyn with Tctex-1 within the population of T hybridoma cells might represent colocalization only at a distinct stage(s) of the cell cycle. To subdivide the cells into distinct stages of cycle, the N17 cells were sorted by DNA content on FACS into G0/G1, S, and G2/M stages before intracellular Ab staining. Although no consistent colocalization was identified in the G0/G1 or S stage populations (data not shown), the G2/M stage population exhibited a unique overlap in staining that was observed in all dividing cells. As shown in Figure 7C, this population is enriched in cells undergoing cytokinesis, which exhibited a striking colocalization of Fyn and Tctex-1 at the developing cleavage furrow that forms as daughter cells begin to divide. In addition, colocalization was evident in these cells at the mitotic spindles. It is interesting to note that Tctex-1, but not Fyn or b-tubulin, seems to interdigitate through the segregated chromosomes and connects the mitotic spindles with the cleavage furrow via fibrous arrays. b-Tubulin staining, although a major component of the mitotic spindles, did not colocalize with Fyn and Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 6. Truncation mapping of Tctex-1 domains that interact with Fyn. The N-terminal domain of Fyn (amino acids 1– 85) was tested as a Gal4 TA fusion protein for interaction capacity with the truncation mutants of Tctex-1 fused to the Gal4 DB domain as assessed by growth on histidine-free medium. Magnitudes of reporter activation are indicated (11 5 strong, 1 5 weak, 2 5 none detected). We have identified and characterized a novel protein interaction between the N-terminal domain of Fyn protein tyrosine kinase and Tctex-1, which has recently been described as a light chain component of the cytoplasmic ATP-dependent dynein motor complex. The interaction was confirmed in vitro using a Tctex-1-GST fusion protein. Tctex-1 can interact with the first 19 amino acids of Fyn, and lysines at positions 7 and 9 of this sequence are critical elements for the interaction. Immunofluorescent confocal microscopy showed that the two proteins consistently colocalize during cytokinesis at both the cleavage furrow and mitotic spindles of a T cell hybridoma, suggesting a role for this interaction in cell division. The ubiquitous expression of Tctex-1 suggests that its interaction with Fyn might also occur in other tissues in which Fyn is readily expressed, most notably neuronal tissues. In view of the limited proliferation of neuronal tissues, the interaction in such tissues would be less likely to play a role in the division of mature cells than in lymphocytes, suggesting additional functional roles for the interaction. Kai et al. (56) have also recently reported tctex-1 as one of several cDNAs that were cloned using a larger portion of Fyn as a bait in the yeast two-hybrid system, although they did not attempt to address the interaction biochemically or further define the interaction domains. Amino acids 1 through 19 of Fyn (see Fig. 5b), which were identified as interacting with Tctex-1, encompass the domain that interacts with unphosphorylated ITAM motifs in lymphocyte Ag receptors (SH4, amino acids 1–10 (31, 32)) and contain three lipid modification sites (Fig. 5b). Cotranslational myristoylation of the glycine at position 2 is believed to be a permanent alteration, while posttranslational palmitoylation of cysteines at positions 3 and 6 is a reversible modification (24 –29, 57). Interestingly, the two lysines at positions 7 and 9 within the Fyn interactive sequence are critical elements for interactions of Fyn with both Tctex-1 (Fig. 5c) and ITAM sequences (32). These basic residues have previously been implicated in enhancing plasma membrane association of Src family kinases by interacting with negatively charged phospholipid head groups (24). Our results and those of Timson Gauen et al. (32) indicate that these lysine residues and presumably other residues in the extreme N-terminal domain of Fyn are also important for protein-protein interactions. One attractive hypothesis would be that a lymphocyte activation or cell cycle-related event might promote depalmitoylation of Fyn and thereby release it from the membrane, exposing the domain that interacts with Tctex-1. It is unlikely that the Fyn fusion proteins are palmitoylated in our yeast experiments, since palmitoylation of this sequence is considered to be dependent upon nearby myristoylation in Src family kinases (24, 26, 28), which is not possible with the initiating methionine of Fyn fused to Gal4. Mutation of the two lysines of Fyn has been shown to alter its localization from the plasma membrane to the cytoplasm (32), although our mutational studies indicate that this localization is not due to Tctex-1 binding (Fig. 5c). The colocalization of Fyn with Tctex-1 during cytokinesis is a particularly intriguing result in light of other recently published The Journal of Immunology 1735 FIGURE 7. Intracellular localization of Tctex-1 and colocalization with Fyn during cytokinesis. A, COS-7 cells were transfected with C-FLAGTctex-1 and intracellular staining was performed with anti-FLAG Ab to visualize Tctex-1 (FITC; green) and b-tubulin (Texas Red). B, Fyn and Tctex-1 colocalize in a subpopulation of N17 T cell hybridomas. The N17 cell line was double-stained with anti-Tctex-1 pAb (Oregon Green 488) and anti-Fyn mAb (Texas Red). The majority of cells demonstrate segre- gated plasma membrane staining of Fyn and perinuclear staining of Tctex-1, and colocalization of the two proteins was evident only in a subpopulation of cells as visualized in yellow (filled arrowheads), while some cells contained distinct cytoplasmic foci of Fyn at sites that are not enriched in Tctex-1 (red; outlined arrowheads). C, Fyn and Tctex-1 colocalize in the cleavage furrow and at the mitotic spindles of N17 T cell hybridomas undergoing cytokinesis. N17 cells in G2/M phase of the cell cycle were sorted by FACS and double-stained with either Oregon Green-labeled anti-Tctex-1 pAb or anti-Fyn pAb plus Texas Red-labeled anti-b-tubulin mAb or anti-Fyn mAb. Each horizontal series shows separate green and red channels of the indicated staining, and both channels merged in the same focal slice from individual cells. The bottom three cells demonstrate colocalization (yellow in the merged image) of Tctex-1 and Fyn at the cleavage furrow (open arrowheads) and mitotic spindles (closed arrowheads). These cells are characteristic of anaphase to early telophase, since they are all beginning to pinch into separate daughter cells. All cells found at this stage of cytokinesis exhibited this representative colocalization. The bar in each panel denotes 10 mm. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 reports. Of particular interest is a study by Yasunaga et al. (22), which determined that proB cells cultured from fyn-deficient mice grew essentially normally until transferred to defined serum-free conditions, at which point these cells arrested during cytokinesis at telophase. In striking contrast, proB cells from normal animals continued through the cell cycle in these serum-free conditions. Their results indicate that Fyn plays a critical role in cell division, although its requirement can be overcome by serum-derived growth factors that presumably bypass the Fyn deficiency block. The same report also demonstrated the localization of Fyn in the cleavage furrow at anaphase of normal proB cells, which we have reproduced in our studies of a T cell hybridoma. Taken together with this genetic evidence, our results suggest that Tctex-1 might provide the crucial scaffold link that tethers Fyn to cytoskeletal motor structures in lymphoid cells, where it functions during cytokinesis. Previous observations of decreased proliferative capacity of thymocytes from fyn-deficient mice (11, 12) might, in fact, be partially explained by this requirement and further reinforces the importance of this newly identified function for the kinase. Ley et al. (23) have reported that Fyn is almost exclusively localized at the centrosome and mitotic spindles of interphase and mitotic T cells, respectively. Since dynein mediates retrograde transport toward these structures, the association of Fyn with Tctex-1 in the dynein complex could clearly mediate this localization of Fyn in lymphocytes. Although we have observed predominantly plasma membrane localization of Fyn in interphase cells using two different Abs in the murine T cell hybridoma, N17 (and other T lymphomas; data not shown), a subpopulation of cells demonstrated distinct cytoplasmic foci of Fyn staining, some of which colocalized with Tctex-1 (Fig. 7C). Ley et al. used a polyclonal Ab directed to the C-terminal domain of Fyn (23), while our Abs were N-terminal reactive, which may account for the predominantly plasma membrane staining pattern in our studies. Roche et al. (58) have demonstrated a requirement for Src kinases, including Fyn, at an earlier stage of the mitotic cell cycle. They reported increased activity of Fyn and other Src kinases in G2/M phaseblocked fibroblasts. In addition, fibroblasts were arrested before prophase, in the same report, by microinjection of an anti-Fyn/Src/ Yes Ab (the same Ab used by Ley et al.) or a GST fusion protein of the Fyn SH2 domain. Finally, Marie-Cardine et al. (59) reported that T cell activation results in tyrosine phosphorylation of a-tubulin and that this phosphorylated a-tubulin can bind the Fyn SH2 domain. Katagiri et al. (60) have also noted tyrosine phosphorylation of tubulin during monocyte differentiation of HL-60 cells and concomitant association of Fyn and Lyn with tubulin. These 1736 Acknowledgments We thank Drs. Pierre Cosson, Marco Colonna, Ulrich Deuschle, Ed Palmer, Jeff Bluestone, Jean Pieters, and Salvatore Valitutti for valuable advice and comments on the manuscript. We also thank Drs. David Beach, John Cambier, Pierre Chevray, Noemi Fusaki, José Garcı́a-Sanz, Beat Imhof, Marina Cella, Andrius Kazlauskas, Daniel Nathans, Antonius Rolink, Tohru Tezuka, Takeshi Watanabe, and Tadashi Yamamoto for generously providing reagents; Bea Pfeiffer and Hans Spalinger for photography; and Irina Serbodova for generating several plasmid constructs for these studies. References 1. Cooke, M. P., and R. M. Perlmutter. 1989. Expression of a novel form of the fyn proto-oncogene in hematopoietic cells. New Biol. 1:66. 2. Samelson, L. E., A. F. Phillips, E. T. Luong, and R. D. Klausner. 1990. Association of the fyn protein-tyrosine kinase with the T-cell antigen receptor. Proc. Natl. Acad. Sci. USA 87:4358. 3. Clark, M. R., K. S. Campbell, A. Kazlauskas, S. A. 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Davidson, and T. W. Mak. 1992. Profound block in thymocyte development in mice lacking p56lck. Nature 357:161. 14. Groves, T., P. Smiley, M. P. Cooke, K. Forbush, R. M. Perlmutter, and C. J. Guidos. 1996. Fyn can partially substitute for Lck in T lymphocyte development. Immunity 5:417. 15. van Oers, N. S. C., B. Lowin-Kropf, D. Finlay, K. Connolly, and A. Weiss. 1996. ab T cell development is abolished in mice lacking both Lck and Fyn protein tyrosine kinases. Immunity 5:429. 16. Takata, M., H. Sabe, A. Hata, T. Inazu, Y. Homma, T. Nukada, H. Yamamura, and T. Kurosaki. 1994. Tyrosine kinases Lyn and Syk regulate B cell receptorcoupled Ca21 mobilization through distinct pathways. EMBO J. 13:1341. 17. Hedin, K. E., M. W. Appleby, and D. E. Clapham. 1995. Developmental regulation of TCR-CD3-dependent [Ca21] responses of individual normal and pp59fyn-deficient T lymphocytes. Immunology 84:183. 18. Jayaraman, T., K. Ondrias, E. Ondriasov, and A. R. Marks. 1996. 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Distinct intracellular localization of Lck and Fyn protein tyrosine kinases in human T lymphocytes. J. Cell Biol. 125:639. 24. Resh, M. D. 1994. Myristylation and palmitylation of Src family members: the fats of the matter. Cell 76:411. 25. Shenoy-Scaria, A. M., L. K. Timson Gauen, J. Kwong, A. S. Shaw, and D. M. Lublin. 1993. Palmitylation of an amino-terminal cysteine motif of protein tyrosine kinases p56lck and p59fyn mediates interaction with glycosyl-phosphatidylinositol-anchored proteins. Mol. Cell. Biol. 13:6385. 26. Koegl, M., P. Zlatkine, S. C. Ley, S. A. Courtneidge, and A. I. Magee. 1994. Palmitoylation of multiple Src-family kinases at a homologous N-terminal motif. Biochem. J. 303:749. 27. Alland, L., S. M. Peseckis, R. E. Atherton, L. Berthiaume, and M. D. Resh. 1994. Dual myristylation and palmitylation of Src family member p59fyn affects subcellular localization. J. Biol. Chem. 269:16701. 28. Bhatnager, R. S., and J. I. Gordon. 1997. Understanding covalent modifications of proteins by lipids: where cell biology and biophysics mingle. Trends Cell Biol. 7:14. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 results taken together suggest potentially important roles for Fyn recruitment to and phosphorylation of microtubule cytoskeletal elements during cellular activation and mitosis and suggest that Tctex-1 might be an important mediator of the Fyn recruitment. What is the role of this interaction during the cell cycle? Although we can only speculate, colocalization of Fyn with Tctex-1 at the cleavage furrow/mitotic spindles and fyn requirements for cytokinesis in proB cells (22) suggest specific roles for these proteins in the division of lymphocytes. Alternatively, the binding of Fyn to Tctex-1 may occur as a capture mechanism for specific cargo in dynein-mediated protein sorting during mitosis. Our understanding of the complex molecular events occurring at the cleavage furrow is only currently unfolding. The role of actin filaments and associated structures in this process is clear, but microtubules and even dynein appear also to play roles (38, 61). Cytoplasmic dynein has also previously been shown to play roles in centrosome separation, anaphase B spindle elongation, and positioning of mitotic spindles during mitosis (38 – 40). Recruitment of Fyn to dynein by Tctex-1 might affect some or all of these mitotic events. Although Tctex-1 does not seem to serve as a major substrate for Fyn in our in vitro phosphorylation studies (Fig. 4a and direct mixing in in vitro phosphorylation reactions, data not shown), it was nevertheless detectably tyrosine phosphorylated. Other proteins within the dynein complex may, however, be more efficacious substrates once the kinase is recruited. Ley et al. (23) have described protein tyrosine phosphorylation surrounding the Fyn at microtubule organizing centers, and as previously mentioned, a-tubulin is a potential tyrosine phosphorylated substrate for Fyn at this location (59, 60). Phosphorylation events identified to date during cytokinesis have predominantly focused on the myosin chains, which undergo serine/threonine phosphorylation (reviewed in Ref. 61). Components of the cytoplasmic dynein complex have been reported to be phosphorylated during cell cycle and transport processes, but again, only serine and threonine phosphorylation has been identified (47, 62– 64). Karki et al. (65) recently reported the association of casein kinase II with dynein, which appears to mediate some of this protein phosphorylation and thereby appears to affect function of the motor complex. Determination of the functional roles of recruited Fyn during cytokinesis in lymphocytes and the possibility of consequent tyrosine phosphorylation of the dynein complex and associated structures should allow for many enlightening future investigations. In summary, accumulating evidence is implicating Fyn kinase as an important effector within the cytoskeleton of lymphocytes, particularly during mitosis. The identification of a direct interaction of the Fyn N-terminal domain with the cytoplasmic dynein motor complex light chain, Tctex-1, provides a novel mechanism for the recruitment of Fyn to this distinct intracellular location. Note Added in Proof. Two potential Fyn substrates that might be important during cytokinesis are the cleavage furrow-associated protein PSTPIP (66) and the inositol 1,4,5-trisphosphate receptor (67). Fyn INTERACTS WITH THE DYNEIN LIGHT CHAIN, Tctex-1 The Journal of Immunology 48. Chevray, P. M., and D. Nathans. 1992. 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