RAFTK, a Novel Member of the Focal Adhesion Kinase Family, Is Phosphorylated and Associates with Signaling Molecules upon Activation of Mature T Lymphocytes

By Ramesh K. Ganju,* William C. Hatch,* Hava Avraham,* Mel A. Ona,* Brian Druker,Dagger Shalom Avraham,* and Jerome E. Groopman*

From the * Divisions of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center,  West Campus, Harvard Institutes of Medicine, Harvard Medical School, Boston, Massachusetts 02215; and the Dagger  Division of Hematology/Oncology, Oregon Health Sciences Center, Portland, Oregon 97201

Summary
Materials and Methods
Results
Discussion
Footnotes
Acknowledgements
References


Summary

The related adhesion focal tyrosine kinase (RAFTK), a recently discovered member of the focal adhesion kinase family, has previously been reported to participate in signal transduction in neuronal cells, megakaryocytes, and B lymphocytes. We have found that RAFTK is constitutively expressed in human T cells and is rapidly phosphorylated upon the activation of the T cell receptor (TCR). This activation also results in an increase in the autophosphorylation and kinase activity of RAFTK. After its stimulation, there was an increase in the association of the src cytoplasmic tyrosine kinase Fyn and the adapter protein Grb2. This association was mediated through the SH2 domains of Fyn and Grb2. RAFTK also co-immunoprecipitates with the SH2 domain of Lck and with the cytoskeletal protein paxillin through its COOH-terminal proline-rich domain. The tyrosine phosphorylation of RAFTK after T cell receptor-mediated stimulation was reduced by the pretreatment of cells with cytochalasin D, suggesting the role of the cytoskeleton in this process. These observations indicate that RAFTK participates in T cell receptor signaling and may act to link signals from the cell surface to the cytoskeleton and thereby affect the host immune response.


Transduction of signals from cell surface receptors involves the activation of numerous molecules having catalytic activity related to phosphorylation. In the case of signaling after the ligation of the TCR/CD3, there is the associated release of calcium, activation of intracytoplasmic protein tyrosine kinases including Fyn and Lck, modulation in phosphatidylinositol metabolism with phosphorylation of PLC-gamma , and changes in the Ras pathway (1). Associated with these activation events, there are changes within the cytoplasm that include alterations in the cytoskeleton (14).

Although considerable knowledge has been generated on such pathways in the T cell response to antigen, there is still much to be learned, particularly regarding the interactions of kinase signaling molecules with the cytoskeletal proteins. One recent study revealed that activation via TCR/CD3 led to important associations and changes in the actin cytoskeleton (17).

We and others have recently identified a novel signaling molecule known as related adhesion focal tyrosine kinase (RAFTK)1, Pyk2, or CAK-beta (18). This molecule is most closely related to the focal adhesion kinase (FAK). FAK has been shown to associate with several signaling molecules and cytoskeletal structures that form in the socalled focal adhesions upon cell attachment to extracellular substrates (21). RAFTK has considerable deduced amino acid and structural similarity with FAK, including consensus motifs in the central catalytic domain, the absence of a transmembrane region, myristylation sites, and SH2 and SH3 domains. Also, similar to FAK, RAFTK has a prolinerich region in the COOH-terminal domain (18).

We have observed that RAFTK is phosphorylated in response to the activation of certain integrins in B lymphocytes (25) and megakaryocytes (26). The induced phosphorylation of RAFTK via calcium-mediated ion channel pathways and after stress activation has been shown in PC12 pheochromocytoma cells (19, 27, 28).

We report the finding that RAFTK is expressed in human T lymphocytes and participates in signaling events triggered by the ligation of the TCR/CD3 complex. We have characterized several of the interacting molecules that associate with RAFTK in human T cells, including the cytoskeletal protein paxillin. These studies provide new information regarding the regulation of pathways of T cell receptor signaling and the associated cytoskeletal changes that may mediate the cellular immune response.


Materials and Methods

Cells and Cell Cultures. The permanent human T cell lines Jurkat and H9 were obtained from the American Type Culture Collection (Rockville, MD) and shown to be mycoplasma-free before their expansion in culture. The cells were carried in DME with 10% FCS, 2 mM glutamine, 50 µg/ml penicillin, and 50 µg/ ml streptomycin. Primary human PBLs were obtained from normal volunteers after obtaining their informed consent and then isolated by Ficoll Hypaque density centrifugation as previously described (29). The anti-CD3-producing hybridoma (OKT-3) was obtained from the American Type Culture Collection and grown in IMDM with 20% FCS. For antibody production, cells were grown in serum- and protein-free hybridoma medium (Sigma Chemical Co., St. Louis, MO) containing Nutridoma-HU 1% (Boehringer Mannheim, Indianapolis, IN).

Reagents and Materials. The lectin PHA was obtained from Pharmacia Biotech (Piscataway, NJ) and the nitrocellulose membrane from BioRad Laboratories (Hercules, CA). The anti-CD3 antibody X35 was obtained from Immunotech (Westbrook, ME), and OKT-3 was purified from OKT-3-producing hybridoma supernatants on protein A-Sepharose columns. Antibodies to Fyn were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and Transduction Laboratories (Lexington, KY). Anti-paxillin antibody was obtained from ICN Biomedicals, Inc. (Costa Mesa, CA). Specific polyclonal antibodies to RAFTK were generated by immunizing New Zealand white rabbits with a bacterially expressed fusion protein consisting of GST and the COOH terminus (amino acids 681-1,009) of the human RAFTK cDNA subcloned into the pGEX-2T expression vector as described (26). The sera were titered against the GST-RAFTK COOH-terminal fusion protein by ELISA, and the serum (R-4250) that revealed the highest titer was used in the subsequent experiments. This antiserum was shown to be specific and not cross-reactive with FAK in earlier experiments (26). Electrophoresis reagents were obtained from BioRad Laboratories. The protease inhibitors leupeptin, aprotinin, and alpha 1 antitrypsin and all other reagents were obtained from Sigma Chemical Co.

Stimulation of Cells. Cells were washed twice with HBSS, (GIBCO BRL, Gaithersburg, MD) and resuspended at 5 × 106 cells/ml in DME. Cells were stimulated with either PHA (10 µg/ ml), alpha -CD3 antibodies ×35 (10 µg/ml), or OKT-3 (10 µg/ml) at 37°C for various time periods. In some experiments, cells were pretreated with EGTA (5 mM) for 5 min at 4°C or cytochalasin D (2 µM) for 60 min at 37°C before stimulation. After stimulation, 2 × 107 cells were microfuged for 10 s and lysed in 1 ml of modified radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM PMSF, 10 µg/ml of aprotinin, leupeptin, and pepstatin, 10 mM sodium vanadate, 10 mM sodium fluoride, and 10 mM sodium pyrophosphate). Total cell lysates were clarified by centrifugation at 10,000 g for 10 min. Protein concentrations were determined by protein assay (BioRad Laboratories).

Immunoprecipitation and Western Blot Analysis. For immunoprecipitation studies, identical amounts of protein from each sample were clarified by incubation with protein A-Sepharose CL-4B (Pharmacia Biotech) for 1 h at 4°C. After the removal of protein A-Sepharose by brief centrifugation, the solution was incubated with different primary antibodies as detailed below for each experiment for 4 h or overnight at 4°C. Immunoprecipitations of the antibody-antigen complexes were performed by incubation for 2 h at 4°C with 75 µl of protein A-Sepharose (10% suspension). Nonspecific bound proteins were removed by washing the Sepharose beads three times with modified RIPA buffer and one time with PBS. Bound proteins were solubilized in 40 µl of 2× Laemmli buffer and further analyzed by immunoblotting. Samples were separated on 7.5% SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk protein and probed with primary antibody for 3 h at room temperature (RT) or 4°C overnight. Immunoreactive bands were visualized using HRP-conjugated secondary antibody and the enhanced chemiluminescent system (Amersham Corp., Arlington Heights, IL). Monoclonal antibody (4G10, IgG2a) was used for Western blot analysis of phosphotyrosine protein.

Glutathione S Transferase Fusion Protein Binding Studies. The RAFTK COOH-terminal domain (amino acids 681 through 1,009) Glutathione S transferase (GST) fusion protein was amplified by the PCR technique and cloned into the pGEX-2T expression vector (Pharmacia Biotech) as previously described (26). The GST- fusion protein was produced by 1 mM isopropyl beta -thiogalactopyranoside induction and purified on a Glutathione-Sepharose column by affinity chromatography according to manufacturer's recommendations (Pharmacia Biotech). GST-fusion protein Grb2SH3 NH2-terminal domain, Grb2-SH3 COOH-terminal domain, Grb2-SH2 domain, and Fyn-SH2 and -SH3 domains were purchased from Santa Cruz Biotechnology. For the binding experiments, 1 mg of cell lysate was mixed with 5 µg of GST-fusion protein and incubated for 1 h at 4°C on a rotatory shaker. GST protein (Santa Cruz Biotechnology) was used as control. 50 µl of Glutathione-Sepharose 4B beads (Pharmacia Biotech) were added to preabsorb the complex. After incubation for 3 h at 4°C on a rotatory shaker, the beads were centrifuged and washed three times with modified RIPA buffer. The bound proteins were eluted by boiling in Laemmli sample buffer and subjected to 7.5% SDSPAGE and Western blot analysis.

Kinase Assays. The immunoprecipitated complexes obtained by immunoprecipitating cell lysates with RAFTK antiserum were washed twice with RIPA buffer and once in kinase buffer (20 mM Hepes, pH 7.4, 50 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 100 mM Na3VO4, and 5 µM ATP). For in vitro kinase assays, the immune complex was incubated in kinase buffer containing 25 µg of poly (Glu/Tyr, 4:1; 20-50 kD; Sigma Chemical Co.) and 5 µCi [32P]ATP at RT for 30 min.

Autophosphorylation Assay. This assay was carried out by incubating the immune complex in a kinase buffer containing 5 µCi [32P]ATP at RT for 30 min. The reaction was stopped by adding 4× SDS sample buffer and by boiling the sample for 5 min. Proteins were then separated on SDS-PAGE and detected by autoradiography.


Results

RAFTK Is Expressed in Human T Lymphocytes and Is Phosphorylated upon T Cell Activation.

To further characterize the signaling pathways in human T cells involved in the immune response, we used as models two permanent T cell lines, Jurkat and H9, as well as primary human PBLs. Analysis by immunoblotting (data not shown) or immunoprecipitation revealed abundant RAFTK protein in these T cells (Fig. 1).



Fig. 1. Tyrosine phosphorylation of RAFTK by T cell receptor cross-linking in Jurkat and H9 human T cell lines or primary human PBLs. Cells were lysed in RIPA buffer. Lysates obtained from 2 × 107 unstimulated (-), alpha -CD3-stimulated or PHA-stimulated Jurkat cells (A), H9 cells (B), or primary human PBLs (C) were immunoprecipitated with RAFTK polyclonal antibody. Immunoprecipitates were size-fractionated on 7.5% SDS-PAGE gels, transferred to nitrocellulose membranes, and then subjected to serial immunoblotting with anti-phosphotyrosine antibody (4G10; top) and anti-RAFTK antibody (bottom). Normal rabbit serum (NRS) was used as a negative control. TCL, total cell lysates.
[View Larger Versions of these Images (19 + 31K GIF file)]

The stimulation of human T cell lines with T cell receptor ligation induces the tyrosine phosphorylation of a phosphoprotein around 115 kD (5, 7). We therefore investigated whether various stimuli associated with such T cell activation modulated the phosphorylation of RAFTK, which has a deduced molecular weight of ~120 kD. As seen in Fig. 1, A and B, an increase in the tyrosine phosphorylation of RAFTK could be specifically observed in the T cell lines Jurkat or H9 after T cell receptor ligation or treatment with the lectin PHA. The membrane was then stripped and reprobed with anti-RAFTK antibody to confirm that equivalent amounts of RAFTK were loaded in each lane (Fig. 1, A and B, bottom). Stimulation of primary PBLs with anti-T cell receptor antibody also induced an increase in the tyrosine phosphorylation of RAFTK (Fig. 1 C).

To determine the time course of tyrosine phosphorylation of RAFTK, Jurkat cells were stimulated with anti-T cell receptor antibody ×35, OKT-3, or with the lectin PHA. Ligation of the TCR/CD3 by monoclonal antibody ×35 or OKT-3 reached a maximum by 2.5-5 min, and declined thereafter (Fig. 2, A and B). PHA stimulation resulted in an increased tyrosine phosphorylation by 5 min that declined slightly thereafter with substantial phosphorylation still detectable at 20 min (Fig. 2 C). Anti-RAFTK immunoblotting of the anti-RAFTK immunoprecipitates showed that the ~115-kD phosphotyrosine polypeptide corresponds to the RAFTK protein (Fig. 2, A-C, bottom). Depending on the resolution of the gels, RAFTK was seen to migrate either as a single band or as a doublet (Figs. 1 and 2).



Fig. 2. Time course of tyrosine phosphorylation of RAFTK upon T cell activation. Jurkat cells (5 × 106/ml) were incubated at 37°C for different time periods with either (A) anti-CD3 antibodies ×35 (10 µg/ml), (B) OKT-3 (10 µg/ml), or (C) PHA (10 µg/ml). Stimulated or unstimulated cells were lysed in RIPA buffer, immunoprecipitated with anti-RAFTK antibody, resolved on SDS-PAGE gels, and subjected to serial immunoblotting with anti-phosphotyrosine antibody (top) and RAFTK antibody (bottom).
[View Larger Versions of these Images (22 + 42K GIF file)]

T Cell Receptor Stimulation Results in Increased RAFTK Autophosphorylation and Kinase Activity.

The autophosphorylation and kinase activities of protein tyrosine kinases can be activated upon their tyrosine phosphorylation, which is essential for their role in signal transduction. We therefore performed an autophosphorylation assay and an in vitro kinase assay in which poly (Glu/Tyr, 4:1) was used as an exogenous substrate to determine the intrinsic tyrosine kinase activity of RAFTK. As shown, T cell receptor stimulation resulted in an increase in the autophosphorylating (Fig. 3 A) as well as the kinase activity of RAFTK (Fig. 3 B). In the autophosphorylation assays, maximal activity was observed at ~2.5-5 min. However, for the in vitro kinase assays, maximal activity was observed at ~5-10 min. The different kinetics observed in autophosphorylation versus in vitro kinase assay could be attributed to the differences in the assays used. The in vitro kinase activity observed in the RAFTK immune complexes using exogenous substrate could be the result of RAFTK and other co-associated kinases such as Fyn.


Fig. 3. RAFTK activation after T cell receptor stimulation. Unstimulated or stimulated Jurkat cell lysates were immunoprecipitated with RAFTK antibody. The immune complexes were either subjected to autophosphorylating activity (A) or in vitro kinase assays, using poly (Glu/ Tyr, 4:1) substrate (B). The 32P-incorporated proteins were resolved on 7.5% SDS-PAGE followed by autoradiography.
[View Larger Version of this Image (59K GIF file)]

RAFTK Associates with the Signaling Molecules Fyn and Grb2.

To further characterize the role that RAFTK might play in T cell signaling after its activation via TCR/ CD3 ligation, we performed immunoprecipitation studies followed by immunoblotting. We observed a specific association of RAFTK with Fyn, a Src family kinase that is known to be capable of associating with TCR (12, 30). A small fraction of Fyn was readily detected as associating with RAFTK before the TCR/CD3 activation of Jurkat cells, and this association increased after their stimulation (Fig. 4 A).



Fig. 4. Association of RAFTK with Fyn and the SH2 domain of Lck. (A) Jurkat cells unstimulated or stimulated with anti-CD3 antibody OKT-3 (10 µg/ml) for different time periods were lysed in RIPA buffer and immunoprecipitated with RAFTK antibody. Immunoprecipitated complexes were resolved on 7.5% SDS-PAGE and immunoblotted with either anti-Fyn antibody (A, top) or RAFTK antibody (A, bottom). (B) Stimulated Jurkat cell lysates were incubated with GST-Fyn SH2 or SH3, or GST-Lck SH2 fusion proteins which were immunoprecipitated with Glutathione beads. The immunoprecipitates were run on 7.5% gels, transferred onto nitrocellulose membranes, and blotted with RAFTK antibody. GST protein only was used as a control.
[View Larger Versions of these Images (37 + 21K GIF file)]

Using GST-fusion proteins corresponding to the SH3 and SH2 domains of Fyn, we observed an association only with the GST-SH2-Fyn protein by immunoprecipitation of the fusion protein followed by immunoblotting with anti-RAFTK antibody (Fig. 4 B). The SH2 domain of Lck, another Src family member, has recently been shown to play an important role in the initiation of signaling events after TCR stimulation (33). We also examined the association of the GST-SH2 domain of Lck with RAFTK. As shown (Fig. 4 B), RAFTK also co-immunoprecipitates with the GST-SH2 domain of Lck.

We then examined the ability of RAFTK to form in vitro complexes with another SH2 and SH3 domain-containing protein, Grb2, which has been shown to interact with RAFTK in other cells (19, 26). As shown in Fig. 5 A, RAFTK immunoprecipitates from activated T cell lysates associated with Grb2. To further characterize this interaction, GST-fusion proteins of the RAFTK-COOH-terminal domain, of Grb2-SH2, or of Grb2-SH3 domains were added to the lysates of the stimulated Jurkat cells. The complexes were immunoprecipitated with Glutathione-conjugated beads and the bound proteins were detected by antiGrb2 (Fig. 5 B) or anti-RAFTK immunoblotting (Fig. 5 C). The GST-RAFTK-COOH-terminal domain associated with Grb2 (Fig. 5 B), whereas Sepharose beads containing the GST-Grb2-SH2 domain bound RAFTK from the activated T cell lysates (Fig. 5 C). Beads containing only GST, GST-NH2-terminal Grb2-SH3, or GST-COOH-terminal Grb2-SH3 failed to bind RAFTK, indicating the specificity of these interactions (Fig. 5 C). These data clearly indicate that the stable interaction between RAFTK and the SH2 domain of Fyn or the SH2 domain of Grb2 can be mimicked in vitro and provide additional evidence that the SH2 domain of these signaling molecules appears to be the principal determinant of RAFTK binding in vivo.



Fig. 5. Association of RAFTK and the adapter protein Grb2 after T cell activation. Cell lysates from Jurkat cells unstimulated or stimulated with anti-CD3 antibody for different time periods were immunoprecipitated with RAFTK antibody. (A) The immune complexes were resolved on 12.5% SDS-PAGE and immunoblotted with anti-Grb2 antibody. (B) Cell lysates were immunoprecipitated with the COOH terminus of RAFTK GST-fusion protein, run on 12.5% SDS-PAGE and blotted with Grb2 antibody. (C) The cell lysates were immunoprecipitated with the SH3 and SH2 domains of Grb2 and blotted with RAFTK antibody.
[View Larger Versions of these Images (21 + 22 + 22K GIF file)]

RAFTK Associates with the Cytoskeletal Protein Paxillin.

After our observation that RAFTK may be co-immunoprecipitated with molecules previously characterized as components of the TCR signaling pathways, we wished to determine whether certain cytoskeletal molecules in T cells may also associate with this novel kinase. Using specific antibodies to RAFTK or paxillin, we found a constitutive association of these two molecules (Fig. 6 A). Using the GST-COOH-terminal RAFTK protein, we observed a modestly increased association with paxillin after TCR/ CD3 stimulation with anti-CD3 antibody (Fig. 6 B).


Fig. 6. Association of RAFTK with the cytoskeletal protein paxillin. (A) Unstimulated or stimulated Jurkat cell lysates were immunoprecipitated with RAFTK antibody, separated on 7.5% gels, and immunoblotted with paxillin antibody. (B) In other experiments, the GST RAFTK COOH-terminal domain protein or GST protein alone as a control was added to unstimulated or stimulated Jurkat cell lysates and immunoprecipitated with Glutathione-Sepharose beads followed by immunoblotting with anti- paxillin antibody.
[View Larger Version of this Image (29K GIF file)]

RAFTK Phosphorylation Is Inhibited by EGTA and Cytochalasin D.

To examine the effect of calcium influx on TCR- induced activation of RAFTK, we treated Jurkat cells with anti-CD3 antibody in the presence or absence of the calcium chelator EGTA. The TCR-induced phosphorylation of RAFTK was partially reduced in the presence of EGTA (Fig. 7 A). This result suggests that the phosphorylation of RAFTK can be mediated in part by the increase in intracellular calcium concentration that is mediated by these stimuli.


Fig. 7. TCR-stimulated tyrosine phosphorylation of RAFTK partly requires calcium and an intact cytoskeleton. Jurkat cells were preincubated with medium plus EGTA (5 mM) or medium alone for 5 min at 4°C (A). Similarly, cells were also preincubated with cytochalasin D (2 µM; +CD) or medium alone (-CD) for 60 min at 37°C (B). Unstimulated cells (-) or cells stimulated for different time periods at 37°C with antiCD3 monoclonal antibody were lysed and immunoprecipitated with RAFTK antibody. The immunoprecipitated complexes were resolved on 7.5% SDS-PAGE and immunoblotted with anti-phosphotyrosine antibody (top), and then stripped and reblotted with antiserum to RAFTK (bottom).
[View Larger Version of this Image (42K GIF file)]

To further investigate the role of the cytoskeleton in the tyrosine phosphorylation of RAFTK, Jurkat T cells were preincubated for 60 min at 37°C with media alone (- CD) or with media plus cytochalasin D (+ CD) before TCR stimulation. Cytochalasin D is known to disrupt the cytoskeletal structure in T lymphocytes and other cells (14, 17, 34). The phosphorylation of RAFTK was reduced after the cytochalasin D treatment of cells (Fig. 7 B). No change in the levels of RAFTK proteins was observed under these conditions (Fig. 7 B). These results suggest that an intact cytoskeleton is important in RAFTK phosphorylation after TCR stimulation.


Discussion

Our studies indicate that RAFTK, a novel signaling molecule that appears to be a member of the FAK family, is present in human T lymphocytes and participates in signaling pathways after T cell activation. After the ligation of the TCR/CD3, there was a robust phosphorylation of RAFTK in both the model permanent T cell lines Jurkat and H9, as well as in primary human PBLs. Furthermore, RAFTK was also activated, which was apparent both in the enhanced autophosphorylation of RAFTK as well as the increase in its in vitro kinase activity as determined using a poly (Glu/Tyr, 1:4) substrate. Parallel studies using other T cell activators, specifically the lectin PHA, revealed a similar phosphorylation of RAFTK in a time- and concentration-dependent manner. Furthermore, it appears that calcium influx may be required for the TCR-mediated tyrosine phosphorylation of RAFTK, as depletion of extracellular calcium by EGTA partially blocked the RAFTK phosphorylation induced by these stimuli. Pyk2/RAFTK phosphorylation has also been shown to be associated with changes in calcium in other cell types (19, 27, 28).

It is noteworthy that after phosphorylation, RAFTK was found to be associated with several well-characterized components of the TCR/CD3 signaling pathways, including Fyn and Grb2. Fyn is a Src family tyrosine kinase known to be capable of associating with the TCR/CD3 complex, and is also believed to play an important role in initiating the changes in phosphorylation that lead to further downstream signaling. This role has been most clearly demonstrated in studies showing the impaired development of CD4+ CD8+ thymocytes from double mutant mice rendered null for Fyn and FAK through homologous recombination (35). In addition, thymocytes from transgenic mice overexpressing Fyn were hyperstimulatable, and overexpression of a catalytically inactive form of Fyn substantially inhibited TCR-mediated activation in otherwise normal thymocytes (36). Grb2 is a well-characterized adapter molecule that is capable of associating with a number of kinases and substrates and may also act to facilitate signaling through the enhancement of the physical association of such partners in enzymatic reactions (37). Grb2 and Shc have also been shown to play an important role in T cell signaling (5, 40- 42). Recently, in PC12 pheochromocytoma cells, Pyk2/ RAFTK has been shown to associate with activated src kinase. This interaction occurred through the SH2 domain of src, which bound to the COOH terminus of Pyk2/ RAFTK (28). The SH2 domain of Fyn has also been shown to associate with RAFTK in other cell types (26). Also in PC12 cells, Grb2 has been reported to be associated with Pyk2/RAFTK (19). Thus, there appears to be similarities in the associations of activated RAFTK with certain signaling molecules in different cell types. RAFTK also associates with the SH2 domain of Lck, which has been shown to play an important role in TCR-induced signal transduction (33).

Our observations on RAFTK suggest that this novel signaling molecule could play a variety of roles in the transduction of T cell signals. Although further work is required to elucidate such functions of RAFTK, the association of RAFTK with the cytoskeletal protein paxillin provides a direction for these future efforts. The confluence of signaling molecules and cytoskeletal components may provide a platform for the regulated interactions of kinases and substrates and may lead to important changes in cell morphology that enable other aspects of the immune response such as adhesion or migration. Work in adherent mesenchymal cells indicates that the formation of the so-called focal adhesions may facilitate the creation of these platforms and mediate cell attachment and transduction of signals (23, 24, 43). Relatively little is known about similar mechanisms in hematopoietic cells like T lymphocytes. Recently, another member of the FAK family, termed FAK B, was identified. Initial studies indicated that FAK B may associate with ZAP-70, an intracytoplasmic protein tyrosine kinase also capable of associating with TCR (44). Amino acid sequence analysis demonstrates that RAFTK is not FAK B; however, future studies of the possible association of RAFTK with FAK B or with stimulatory molecules like ZAP-70 will be of value in elucidating these signaling mechanisms.

There is relatively limited information available on the convergence of protein tyrosine kinases and cytoskeletal elements in T lymphocytes. Several T cell surface structures, including CD11a/CD18 and CD44, associate with the cytoskeleton upon receptor cross-linking. Recently, the interaction of the zeta  chain of TCR with the actin cytoskeleton upon T cell activation was demonstrated (17). Our results revealed that RAFTK co-associates with paxillin, a major component of the cytoskeleton. Like FAK, the proline-rich COOH terminus of RAFTK binds to paxillin (24). Furthermore, the pretreatment of cells with cytochalasin D results in the reduced tyrosine phosphorylation of RAFTK upon T cell receptor activation. This result suggests that RAFTK phosphorylation may require the formation of a cytoskeletal complex, which provides a foundation for the interactions and compartmentalization of kinases and substrates. Future studies will further define whether other cytoskeletal components are associated with RAFTK upon T cell activation.

It has been suggested that in addition to FAK B, FAK may also participate in regulatory events after T lymphocyte activation (45). These earlier data, in conjunction with the results from our study, strongly suggest that molecules like FAK and RAFTK are important in coordinating a repertoire of kinases and adapter molecules in T cells, which then are capable of transmitting regulated signals downstream to the transcriptional activators that modulate gene expression. In this regard, in the PC12 neuronal cell model, Pyk2/ RAFTK activation coupled stress signals to the c-Jun-NH2 terminal kinase pathway (27). This pathway is known to activate AP-1 and has been previously reported to participate in T lymphocyte signaling after costimulation (46, 47). Our future focus will be to assess whether inherited or acquired abnormalities in T cell function involve the activation and subsequent associative functions of RAFTK as it colocalizes signaling molecules with the cytoskeleton, and how this may be linked to transcriptional activation.


Footnotes

Address correspondence to Dr. Jerome Groopman, Divisions of Experimental Medicine and Hematology/ Oncology, Beth Israel Deaconess Medical Center, West Campus, Harvard Institutes of Medicine, Harvard Medical School, 1 Deaconess Rd., Boston, MA 02215.

Received for publication 4 November 1996 and in revised form 15 January 1997.

   1Abbreviations used in this paper: FAK, focal adhesion kinase; GST, Glutathione S transferase; RAFTK, related adhesion focal tyrosine kinase; RIPA, radioimmunoprecipitation assay; RT, room temperature.

We thank our colleagues Zhong-Ying Liu and Jian-Feng Wang for their technical assistance. We would also like to thank Dr. David J. McConkey and Dr. Dananagoud Hiregowdara for their helpful discussions. We are grateful to Janet Delahanty for her editing and preparation of the figures and to Jennifer McGrath and Evelyn Gould for their assistance with the figures for this manuscript. Finally, we appreciate Tee Trac for her typing assistance and Delroy Heath for facilitating our receipt of the needed reagents for the experiments. This paper is dedicated to the memory of our colleague Dr. Dananagoud Hiregowdara.

This work was supported in part by National Institutes of Health grants HL 53745-02, HL 43510-07, HL 55187-01, HL 51456-02, and HL 55445-01, and the David Geffen Foundation.


References

1. Chan, A.C., B.A. Irving, and A. Weiss. 1992. New insights into T-cell antigen receptor structure and signal transduction. Curr. Opin. Immunol. 4: 246-251 [Medline].
2. Donovan, J.A., R.L. Wange, W.Y. Langdon, and L.E. Samelson. 1994. The protein product of the c-cbl protooncogene is the 120 Kd tyrosine-phosphorylated protein in Jurkat cells activated via the T cell antigen receptor. J. Biol. Chem. 269: 22921-22924 [Abstract/Free Full Text].
3. Exley, M., L. Varticovski, M. Peter, J. Sancho, and C. Terhorst. 1994. Association of phosphatidylinositol 3-kinase with a specific sequence of the T cell receptor zeta chain is dependent on T cell activation. J. Biol. Chem. 269: 15140-15146 [Abstract/Free Full Text].
4. Fukazawa, T., K.A. Reedquist, G. Panchamoorthy, S. Soltoff, T. Trub, B. Druker, L. Cantley, S.E. Shoelson, and H. Band. 1995. T cell activation-dependent association between the p85 subunit of the phosphatidylinositol 3-kinase and Grb2/ phospholipase C-gamma 1-binding phosphotyrosyl protein pp36/38. J. Biol. Chem. 270: 20177-20182 [Abstract/Free Full Text].
5. Motto, D.G., S.E. Ross, J.K. Jackman, Q. Sun, A.L. Olson, P.R. Findell, and G.A. Koretzky. 1994. In vivo association of Grb2 with pp116, a substrate of the T cell antigen receptoractivated protein tyrosine kinase. J. Biol. Chem. 269: 21608-21613 [Abstract/Free Full Text].
6. Wange, R.L., and L.E. Samelson. 1996. Complex complexes: signaling at the TCR. Immunity. 5: 197-205 [Medline].
7. Hsi, E.D., J.N. Siegel, Y. Minami, E.T. Luong, R.D. Klausner, and L.E. Samelson. 1988. T cell activation induces rapid tyrosine phosphorylation of a limited number of cellular substrates. J. Biol. Chem. 264: 10836-10842 [Abstract/Free Full Text].
8. Janeway, C.A., Jr, and K. Bottomly. 1994. Signals and signs for lymphocyte responses. Cell. 76: 275-285 [Medline].
9. Klausner, R.D., and L.E. Samelson. 1996. T cell antigen receptor activation pathways: the tyrosine kinase connection. Cell. 64: 875-878 .
10. Nel, A.E., S. Gupta, L. Lee, J.A. Ledbetter, and S.B. Kanner. 1995. Ligation of the T-cell antigen receptor (TCR) induces association of hSos1, ZAP-70, phospholipase C-gamma 1, and other phosphoproteins with Grb2 and the zeta-chain of the TCR. J. Biol. Chem. 270: 18428-18436 [Abstract/Free Full Text].
11. Straus, D.B., and A. Weiss. 1992. Genetic evidence for the involvement of the Lck tyrosine kinase in signal transduction through the T cell antigen receptor. Cell. 70: 585-593 [Medline].
12. Gauen, L.K.T., A.N. Kong, L.E. Samelson, and A.S. Shaw. 1992. p59fyn tyrosine kinase associates with multiple T-cell receptor subunits through its unique amino-terminal domain. Mol. Cell Biol. 12: 5438-5446 [Abstract].
13. Tsygankov, A.Y., B.M. Broker, J. Fargnoli, J.A. Ledbetter, and J.B. Bolen. 1992. Activation of tyrosine kinase p60fyn following T cell antigen receptor cross-linking. J. Biol. Chem. 267: 18259-18262 [Abstract/Free Full Text].
14. Valitutti, S., M. Dessing, K. Aktories, H. Gallati, and A. Lanzavecchia. 1995. Sustained signaling leading to T cell activation results from prolonged T cell receptor occupancy. Role of T cell actin cytoskeleton. J. Exp. Med. 181: 577-584 [Abstract].
15. Dubois, T., J.P. Oudinet, F. Russo-Marie, and B. Rothhut. 1995. In vivo and in vitro phosphorylation of annexin II in T cells: potential regulation by annexin V.  Biochem. J. 310: 243-248 [Medline].
16. Egerton, M., W.H. Burgess, D. Chen, B.J. Druker, A. Bretscher, and L.E. Samelson. 1992. Identification of ezrin as an 81 Kd tyrosine-phosphorylated protein in T cells. J. Immunol. 149: 1847-1852 [Abstract/Free Full Text].
17. Rozdzial, M.M., B. Malissen, and T.H. Finkel. 1995. Tyrosine-phosphorylated T cell receptor zeta chain associates with the actin cytoskeleton upon activation of mature T lymphocytes. Immunity. 3: 623-633 [Medline].
18. Avraham, S.A., R. London, Y. Fu, S. Ota, D. Hiregowdara, J. Li, S. Jiang, L.M. Pasztor, R.A. White, J.E. Groopman, and H. Avraham. 1995. Identification and characterization of a novel related adhesion focal tyrosine kinase (RAFTK) from megakaryocytes and brain. J. Biol. Chem. 270: 27742-27751 [Abstract/Free Full Text].
19. Lev, S., H. Moreno, R. Martinez, P. Canoll, E. Peles, J.M. Musacchio, G.D. Plowman, B. Rudy, and J. Schlessinger. 1995. Protein tyrosine kinase PYK2 involved in Ca2+-induced regulation of ion channel and MAP kinase functions. Nature (Lond.). 376: 737-745 [Medline].
20. Sasaki, H., K. Nagura, M. Ishino, H. Tobioka, K. Kotani, and T. Sasaki. 1995. Cloning and characterization of cell adhesion kinase beta, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily. J. Biol. Chem. 270: 21206-21219 [Abstract/Free Full Text].
21. Burridge, K., C.E. Turner, and L.H. Romer. 1992. Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J. Cell Biol. 119: 893-903 [Abstract].
22. Hanks, S.K., M.B. Calalb, M.C. Harper, and S.K. Patel. 1992. Focal adhesion protein-tyrosine kinase phosphorylated in response to cell attachment to fibronectin. Proc. Natl. Acad. Sci. USA. 89: 8487-8491 [Abstract].
23. Richardson, A., and J.T. Parsons. 1995. Signal transduction through integrins: a central role for focal adhesion kinase? BioEssays. 17: 229-236 [Medline].
24. Clark, E.A., and J.S. Brugge. 1995. Integrins and signal transduction pathways: the road taken. Science (Wash. DC). 268: 233-239 [Medline].
25. Astier, A., H. Avraham, S.N. Manie, J.E. Groopman, T. Canty, S. Avraham, and A.S. Freedman. 1997. The related adhesion focal tyrosine kinase (RAFTK) is tyrosine phosphorylated after beta 1 integrin stimulation in B cells and binds to p130cas. J. Biol. Chem. 272: 228-232 [Abstract/Free Full Text].
26. Li, J., H. Avraham, R.A. Rogers, S. Raja, and S. Avraham. 1996. Characterization of RAFTK, a novel focal adhesion kinase, and its integrin-dependent phosphorylation and activation in megakaryocytes. Blood. 88: 417-428 [Abstract/Free Full Text].
27. Tokiwa, G., I. Dikic, S. Lev, and J. Schlessinger. 1996. Activation of Pyk2 by stress signals and coupling with JNK signaling pathway. Science (Wash. DC). 273: 792-794 [Abstract].
28. Dikic, I., G. Tokiwa, S. Lev, S.A. Courtneidge, and J. Schlessinger. 1996. A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation. Nature (Lond.). 383: 547-550 [Medline].
29. Boyum, A. 1968. Isolation and removal of lymphocytes from bone marrow of rats and guinea-pigs. Scand. J. Clin. Lab. Invest. 97(Suppl.):91-106.
30. Katagiri, T., K. Urakawa, Y. Yamanashi, K. Semba, T. Takahashi, K. Toyoshima, T. Yamamoto, and K. Kano. 1989. Overexpression of src family gene for tyrosine-kinase p59fyn in CD4-CD8- T cells of mice with a lymphoproliferative disorder. Proc. Natl. Acad. Sci. USA. 86: 10064-10068 [Abstract].
31. Prasad, K.V., O. Janssen, R. Kapeller, M. Raab, L.C. Cantley, and C.E. Rudd. 1993. Src-homology 3 domain of protein kinase p59fyn mediates binding to phosphatidylinositol 3-kinase in T cells. Proc. Natl. Acad. Sci. USA. 90: 7366-7370 [Abstract].
32. 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: 4358-4362 [Abstract].
33. Straus, D.B., A.C. Chan, B. Patai, and A. Weiss. 1996. SH2 domain function is essential for the role of the Lck tyrosine kinase in T-cell receptor signal transduction. J. Biol. Chem. 271: 9976-9981 [Abstract/Free Full Text].
34. Prat, A.G., Y.F. Xiao, D.A. Ausiello, and H.F. Cantiello. 1995. cAMP-independent regulation of CFTR by the actin cytoskeleton. Am. J. Physiol. 268: 1552-1561 .
35. Kanazawa, S., I. Dusko, M. Hashiyama, T. Noumura, T. Yamamoto, T. Suda, and S. Aizawa. 1996. p59fyn-p125FAK cooperation in development of CD4+CD8+ thymocytes. Blood. 87: 865-870 [Abstract/Free Full Text].
36. Cooke, M.P., K.M. Abraham, K.A. Forbush, and R.M. Perlmutter. 1991. Regulation of T cell receptor signaling by a src family protein-tyrosine kinase (p59fyn). Cell. 65: 281-291 [Medline].
37. Li, N., A. Batzer, R. Daly, V. Yajnik, E. Skolnik, P. Chardin, D. Bar-Sagi, B. Margolis, and J. Schlessinger. 1993. Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling. Nature (Lond.). 363: 85-88 [Medline].
38. Koch, C.A., D. Anderson, M.F. Moran, C. Ellis, and T. Pawson. 1991. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science (Wash. DC). 252: 668-674 [Medline].
39. Pawson, T., and G.D. Gish. 1992. SH2 and SH3 domains: from structure to function. Cell. 71: 359-362 [Medline].
40. Meisner, H., B.R. Conway, D. Hartley, and M.P. Czech. 1995. Interactions of Cbl with Grb2 and phosphatidylinositol 3'-kinase in activated Jurkat cells. Mol. Cell Biol. 15: 3571-3578 [Abstract].
41. Fukazawa, T., K.A. Reedquist, T. Trub, S. Soltoff, G. Panchamoorthy, B. Druker, L. Cantley, S.E. Shoelson, and H. Band. 1995. The SH3 domain-binding T cell tyrosyl phosphoprotein p120. Demonstration of its identity with the c-cbl protooncogene product and in vivo complexes with Fyn, Grb2, and phosphatidylinositol 3-kinase. J. Biol. Chem. 270: 19141-19150 [Abstract/Free Full Text].
42. Ravichandran, K.S., K.K. Lee, Z. Songyang, L.C. Cantley, P. Burn, and S.J. Burakoff. 1993. Interaction of Shc with the zeta chain of the T cell receptor upon T cell activation. Science (Wash. DC). 262: 902-905 [Medline].
43. Schlaepfer, D.D., S.K. Hanks, T. Hunter, and P. van der Geer. 1994. Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature (Lond.). 372: 786-791 [Medline].
44. Kanner, S.B., A. Aruffo, and P.Y. Chan. 1994. Lymphocyte antigen receptor activation of a focal adhesion kinase-related tyrosine kinase substrate. Proc. Natl. Acad. Sci. USA. 91: 10484-10487 [Abstract/Free Full Text].
45. Maguire, J.E., K.M. Danahey, L.C. Burkly, and G.A. van Seventer. 1995. T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells. J. Exp. Med. 182: 2079-2090 [Abstract].
46. Su, B., E. Jacinto, M. Hibi, T. Kallunki, M. Karin, and Y. Ben-Neriah. 1994. JNK is involved in signal integration during costimulation of T lymphocytes. Cell. 77: 727-736 [Medline].
47. del Arco, P.G., S. Martinez-Martinez, V. Calvo, A.L. Armesilla, and J.M. Redondo. 1996. JNK (c-Jun NH2-terminal kinase) is a target for antioxidants in T lymphocytes. J. Biol. Chem. 271: 26335-26340 [Abstract/Free Full Text].

Copyright © 1997 by The Rockefeller University Press.