The related adhesion focal tyrosine kinase forms a complex with paxillin in hematopoietic cells.

Related adhesion focal tyrosine kinase (RAFTK), also known as proline-rich tyrosine kinase 2 and cellular adhesion kinase β, has been recently cloned and characterized as a member of the focal adhesion kinase (FAK) subfamily. RAFTK has an overall 48% amino acid homology to p125FAK and contains a kinase domain but lacks a transmembrane region, myristylation sites, and Src homology region 2 and 3 domains. By Northern blot analysis, RAFTK is expressed in myeloid, lymphoid, and megakaryocytic hematopoietic cells. Like p125FAK, we found that RAFTK interacts with the focal adhesion protein paxillin. In the lymphoid cell line BaF3 and the myeloid cell line 32Dcl3, RAFTK coprecipitates with paxillin. Using in vitro binding assays, RAFTK and paxillin were shown to bind directly, through a segment of paxillin that required amino acids 100-227 and a domain in the C terminus of RAFTK. In vitro, RAFTK could phosphorylate paxillin on tyrosine residues. These results suggest that RAFTK, as well as p125FAK, may be important in phosphotyrosine-signaling events within the focal adhesion.

transduction of megakaryocytes and in hematopoietic cells. RAFTK has been shown to be expressed in platelets, CD34 ϩ marrow cells, and primary bone marrow megakaryocytes, as well as in some nonhematopoietic cells, including brain cells (1). Although RAFTK and p125 FAK are structurally similar, it is not currently known whether their functions are also similar.
p125 FAK is involved in integrin signaling, phosphorylates cytoskeletal proteins, and is known to associate with proteins at focal adhesions, the specialized structures in which the actin cytoskeleton is connected to transmembrane integrin molecules (4). One of the proteins that directly interacts with p125 FAK in the focal adhesions is paxillin (5). Paxillin is a substrate for p125 FAK and also serves as a binding site for vinculin, talin, CRK, CRKL, and c-Src (6 -8). Paxillin is believed to be an important substrate and binding site for various oncogene products, such as BCR/ABL, v-Crk, and v-Src (9 -11).
In preliminary studies, it was determined that, like p125 FAK , RAFTK localized in the focal adhesion. Also, RAFTK is expressed in most hematopoietic cells (myeloid, lymphoid, and megakaryocytic). In an effort to compare the function of RAFTK and p125 FAK , we have examined the interaction of RAFTK with paxillin in hematopoietic cells. We show that there is constitutive binding of paxillin to RAFTK and that RAFTK can act as a kinase for paxillin.
Northern Analysis-RNA was isolated from various cells by extraction with guanidine isothiocyanate as described (14). Samples of 15 g of total RNA were subjected to Northern analysis. cDNA for full-length paxillin (9), RAFTK C terminus (15), p125 FAK C terminus (16), and glyceraldehyde-3-phosphate dehydrogenase were used as specific probes.
Preparation of Cell Lysates, Immunoprecipitations, and Immunoblotting-Cells were lysed in lysis buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, and 0.42% NaF) containing inhibitors (10 l of 100 M phenylmethylsulfonyl fluoride, 10 l of 100 M Na 3 VO 4 , 5 l of aprotinin (Sigma), and 2 l of 10 mg/ml leupeptin) (9). Immunoprecipitations were performed with the antipaxillin (clone 5H11, as * This work was supported by National Institutes of Health Grants CA60821 (to R. S.), HL55445 (to S. A.), HL51456 (to H. A.), and CA36167 (to J. D. G.). 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.
Production of Paxillin Monoclonal Antibodies-Mice were immunized by repeated injections of GST-full-length human paxillin fusion protein (amino acids 1-527) emulsified in Freund's complete adjuvant (Sigma) (18). Hybridomas were screened by enzyme-linked immunosorbent assay against the immunizing protein and further by immunoblotting. Positive hybridomas were recloned and isotyped by enzyme-linked immunosorbent assay, and high titer ascites were produced by standard techniques (14). Monoclonal antipaxillin antibodies were purified on protein A-Sepharose beads (Pharmacia Biotech Inc.). Five separate antipaxillin antibodies were identified and characterized with respect to their ability to be used for immunoblotting, immunoprecipitations, and cell staining (data not shown). One hybridoma clone (5H11) was used in this study (IgG1 isotype) and was found to specifically identify paxillin in human and murine cells by immunoblot, immunoprecipitation, and cell staining (data not shown).
GST fusion proteins in the vector pGEX-2TK containing human C-terminal p125 FAK (amino acids 896-1052; courtesy of Dr. C. Morimoto, Dana-Farber Cancer Institute) were expressed and isolated as per previous protocols (16).
Precipitations were performed with 15 g of fusion protein on glutathione beads using the lysate from 20 ϫ 10 6 cell lysates as described (9).
In Vitro Kinase Assay-RAFTK was immunoprecipitated by incubating lysates from Mo7e cells, containing large amounts of RAFTK in the immunoprecipitates and minimal or no paxillin coimmunoprecipitating in this complex, for 90 min at 4°C with anti-RAFTK rabbit polyclonal antibody R4250, along with protein A-Sepharose beads. The complexes were washed with lysis buffer and then resuspended in 20 l of kinase buffer (10 mM HEPES, 3 mM MnCl 2 , pH 7.3) (8). Approximately 15 g of GST-paxillin fusion protein (previously resuspended into the kinase buffer) was added to the mixture with 10 Ci of [␥-32 P]ATP. Following a 20-min incubation period at room temperature, the reactions were terminated by boiling the samples in SDS-PAGE sample buffer. Samples were resolved on a 10% SDS-PAGE gel and then visualized by autoradiography.
Western Blot Binding Assay (Far Western)-Immunoprecipitations were performed using antipaxillin (clone 5H11) mouse monoclonal antibody, and anti-RAFTK (R4250) rabbit polyclonal antibody as described previously and separated on 7.5% SDS-PAGE gels (9). The gels were then transferred as described previously (9). The blots were incubated at 4°C overnight in 5% dry milk and phosphate-buffered saline with 0.1% Tween 20. The blots were washed for 1 h every 15 min at room temperature in phosphate-buffered saline with 0.1% Tween 20 between incubations. The blots were incubated in GST fusion proteins, full-length paxillin, the four tandem LIM domains of paxillin (amino acids 316 -527), and PE8 for 2 h. The blots were washed and placed into an anti-GST mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h. After washing, anti-mouse Ig, horseradish peroxidase (Amersham) was added for 1 h. The membranes were processed per established methods using the enhanced chemiluminescense technique (Amersham) (12).

Expression of RAFTK in Hematopoietic Cell
Lines-To determine the expression of RAFTK in various cell lines, we performed Northern blot analysis. Several hematopoietic, megakaryocytic, and adherent fibroblast cell line RNAs were used to compare the levels of expression of p125 FAK , RAFTK, and paxillin. The hematopoeitic cell lines used were BaF3, 32Dcl3, and K562. The megakaryocytic cell lines used were CMK, DAMI, and Mo7e. The fibroblast cell lines used were CV-1, FS-2, and NIH3T3. The membrane was probed with RAFTK-, p125 FAK -, paxillin-, and glyceraldehyde-3-phosphate dehydrogenase-specific probes sequentially.
Expression of RAFTK was found in megakaryocytic cell lines (Fig. 1). However, in nonhematopoietic cell lines, RAFTK expression was low. In myeloid and lymphoid cell lines 32Dcl3, BaF3, and K562, RAFTK was moderately expressed. Paxillin was expressed in both the hematopoietic and adherent fibroblast cell lines and only minimally in the megakaryocytic cell lines. In contrast, p125 FAK was expressed at relatively consistent levels in the fibroblastic and megakaryocytic cell types tested, but at a somewhat lower level in myeloid and lymphoid cell lines.
RAFTK Coprecipitates with the Focal Adhesion Protein Paxillin-It has been previously shown that paxillin coimmunoprecipitates with p125 FAK in hematopoietic cells (6). Using the BaF3 cell line, RAFTK was found to coimmunoprecipitate with paxillin in unstimulated, IL-3-stimulated, and p210 BCR/ABLtransformed BaF3 cells (Fig. 2, A and B). Identical results were obtained when cell lysates were immunoprecipitated with anti-RAFTK and immunoblotted with paxillin. These results indicate constitutive association of RAFTK and paxillin.
Previously, it has been shown that the C terminus of p125 FAK binds directly to a region in the N terminus of paxillin (19). Since RAFTK is a member of the FAK family, and constitutive association of RAFTK and paxillin was observed, GST fusion protein precipitations were performed to determine which regions of paxillin would associate with RAFTK and vice versa. Lysates from BaF3 cells transformed by the oncogene BCR/ABL were precipitated with various GST fusion proteins of paxillin (full-length, PE7-PE10), RAFTK (C terminus), and p125 FAK (C terminus). The samples were separated by gradient (6 -12%) SDS-PAGE as described under "Materials and Meth- ods." The precipitations revealed that the binding site of the C terminus of RAFTK to paxillin is located within the amino acid region 100 -227 (Figs. 3 and 4), which we have previously identified as a talin binding region in hematopietic cells (9). Thus, RAFTK and p125 FAK both bind paxillin with their C termini.
RAFTK Binds Directly to Paxillin-Previous groups have shown that the C terminus of p125 FAK binds directly to paxillin (5,7,16). We determined whether the same would be true for RAFTK. To show direct binding between the C terminus of RAFTK and paxillin, we performed far Western blotting. Lysates of BaF3 cells were immunoprecipitated with the RAFTK rabbit polyclonal antibody (R4250). The samples were then separated on a 7.5% SDS-PAGE gel and subjected to far Western blotting as described under "Materials and Methods." Fig.  5 shows that the GST protein alone does not bind to RAFTK, nor do the PE8-paxillin-GST fusion protein (putative talin binding region, deletion of amino acids 100 -227) or the LIM domains. Furthermore, the results shown in Fig. 5 demonstrate that the full-length paxillin binds directly to RAFTK and supports the location of the binding region in the C terminus of RAFTK. The experiment was also performed in reverse, with paxillin immunoprecipitation and RAFTK far Western blotting, which reconfirmed the direct binding (data not shown).
Taken together, these experiments demonstrate that RAFTK and paxillin not only coprecipitate with each other but can bind directly to each other.
RAFTK Phosphorylates Paxillin in Vitro-RAFTK contains a catalytic domain, which could potentially phosphorylate paxillin in a similar fashion as p125 FAK . An in vitro phosphorylation experiment using lysates from the megakaryocytic cell line Mo7e was performed, because these cells contain very small amounts of endogenous paxillin. RAFTK immunoprecipitated with the R4250 antibody was incubated in a kinase reaction with various GST fusion proteins of paxillin and was tested for phosphorylation. Full-length paxillin, PE7 (N-terminal region of paxillin without the LIM domains), and the LIM domains were studied. After in vitro phosphorylation of the various paxillin fusion proteins, the samples were resolved on a 10% SDS-PAGE gel, and the results were visualized by autoradiography. The results in Fig. 6 demonstrate that RAFTK phosphorylates the N-terminal half of paxillin and not the LIM domains. In the N-terminal half of paxillin there are six tyrosine residues (at positions 31, 33, 40, 88, 118, and 181; human sequence), which could be potential substrate sites for the RAFTK kinase.

DISCUSSION
The cytoskeleton is essential for many cellular functions, including regulation of cell shape, flexibility, and adhesive properties (20). Part of the cytoskeleton and plasma membrane form a region known as the focal adhesion (21). Focal adhesions are structures that form adherent contacts with the extracellular matrix (21). Extracellular matrix proteins act through receptors known as integrins localized in focal adhesions. The intracellular domains of integrins interact directly with focal adhesion proteins such as tensin and p125 FAK (22). Various events, such as exposure to cytokines and growth factors, can modulate the expression and function of adhesion receptors and the formation of focal adhesions (23). Phosphorylation of proteins within focal adhesions is closely associated with changes in the structure of the actin cytoskeleton. Some proteins contained in the focal adhesion include talin, tensin, vinculin, paxillin, p125 FAK , and ␣-actinin (21). p125 FAK is a unique tyrosine kinase localized to this region and has been shown to phosphorylate various proteins, on tyrosine, in focal adhesions by several extracellular stimuli (24). p125 FAK has been previously shown to be activated by integrin cross-linking, growth factor stimulation, and oncogenic signaling (25). After phosphorylation, p125 FAK is known to associate with several proteins, including paxillin, tensin, Csk, and the Src family kinases (through tyrosine 397 on p125 FAK ) (26). p125 FAK has also been shown to associate with GRB2 in macrophages via tyrosine residue 397 (27). This interaction links p125 FAK to the Ras pathway (28). p125 FAK may be important in the turnover of focal adhesion contacts during cell migration (29). The tyrosine phosphorylation of p125 FAK precedes cell spreading and the formation of focal adhesion contacts in adherent fibroblasts. When cells are detached, p125 FAK tyrosine kinase activity is rapidly reduced.
RAFTK is another protein tyrosine kinase recently cloned out of a megakaryocytic library similar to p125 FAK . RAFTK was localized to chromosome 8 in humans (1) and, like p125 FAK , was shown to become tyrosine-phosphorylated in response to thrombin stimulation (1). Tyrosine residue 402 of RAFTK is similar to that of tyrosine residue 397 of p125 FAK (1). This site has the potential to bind the SH2 domains of Src family kinases.
To identify the role of RAFTK in hematopoietic cells, expression of RAFTK was examined in these cells. RNA from hematopoietic (32Dcl3 and BaF3), megakaryocytic (CMK, DAMI, and Mo7e), and adherent (CV-1, FS-2, and NIH3T3) fibroblasts were tested for expression of RAFTK, p125 FAK , and paxillin. In the hematopoietic cells, the expression levels of p125 FAK and paxillin were high, whereas those of RAFTK were only moderate. Interestingly, in the megakaryocytic cells, expression of RAFTK and p125 FAK were high, but the levels of paxillin were extremely low. But in the adherent fibroblasts, the expression of p125 FAK was high, levels of paxillin were moderate, and the expression levels of RAFTK were very low.
In hematopoietic cells, p125 FAK is known to be expressed, and it associates with one of its major substrates, paxillin (6). Paxillin is a focal adhesion protein known to associate with several tyrosine kinases. Paxillin has also been shown to be important in signaling transduction pathways of hematopoietic cells (8). The results in this study indicate that in addition to p125 FAK , RAFTK and paxillin associate with each other in hematopoietic cells. In megakaryocytes, it has been shown that both RAFTK and paxillin colocalize with each other (data not shown). Like p125 FAK , the C terminus of RAFTK binds directly to the N terminus of paxillin. RAFTK binds in the region of amino acids 100 -227 of paxillin. This is probably a different binding site from that of p125 FAK , which binds in the region of amino acids 51-315 (19). p125 FAK and RAFTK may bind to paxillin in different regions, because they may tyrosine phosphorylate different residues on paxillin. Their binding locations may give them greater access to the tyrosine residues they phosphorylate, and these data may show that p125 FAK and RAFTK phosphorylate different tyrosines on paxillin.
RAFTK (1), cellular adhesion kinase ␤ (3), and proline-rich tyrosine kinase 2 (PYK2) (2) are the same molecules and are cloned via different strategies with interesting findings. The results obtained from studies of RAFTK, cellular adhesion kinase ␤, and PYK2 may offer insight into the function of the FAK family. For example, the tyrosine phosphorylation state of cellular adhesion kinase ␤ was not reduced on trypsinization or enhanced in response to plating 3Y1 cells onto fibronectin (3). PYK2 was isolated when libraries were screened with the GRB2 adaptor protein (2), and it has been shown that calcium influx in PC12 cells will cause rapid tyrosine phosphorylation of PYK2 (2). Also, carbachol, acting through the nicotinic acetylcholine receptor, and bradykinin stimulation will cause tyrosine phosphorylation of PYK2 in PC12 cells (2). These interactions are thought to play a role in the Ras signaling pathway, very similar to p125 FAK (2). Another homologue to p125 FAK was identified as fakB, a 120-kDa tyrosine kinase that is localized in focal adhesions of cells (30). Tyrosine phosphorylation of fakB is rapidly augmented in human B and T cells following antigen receptor cross-linking with antibody (30). fakB is a putative downstream component of antigen receptor signaling in both T and B lymphocytes.
The first substrate for RAFTK has been identified in this FIG. 5. RAFTK binds directly to paxillin. Immunoprecipitates using lysates of unstimulated BaF3 cells (BaF3(Ϫ)), BaF3 cells stimulated with IL-3 (BaF3(ϩ)), and BaF3.p210 BCR/ABL cells with anti-RAFTK antibody (I.P., 20 ϫ 10 6 cells) were processed as described under "Materials and Methods," applied to 7.5% SDS-PAGE, and transferred to Immobilon-P membranes. The membranes were processed for a far Western blot as described under "Materials and Methods" with the various GST fusion proteins: GST, GST-paxillin (full-length), and GST-PE8. Molecular masses are shown in kDa.
FIG. 6. In vitro kinase assay with paxillin. Immunoprecipitates of cell lysates of Mo7e with anti-RAFTK antibody (20 ϫ 10 6 cells) were processed as described under "Materials and Methods." GST-paxillin fusion proteins were added, and the kinase reaction was performed as described under "Materials and Methods" and applied to 10% SDS-PAGE. The fusion proteins used were GST-paxillin (full-length (F.L.), LIM, and PE7. Bands were visualized using autoradiography. Molecular masses are shown in kDa.
report. RAFTK is a tyrosine kinase that can in vitro phosphorylate paxillin on its N-terminal half (amino acids 1-315). Although the exact site is unknown, only six tyrosine residues are present in this area (at positions 31, 33, 40, 88, 118, and 181; human sequence) (9). Several of these sites have been shown to be important in binding to other proteins, such as tyrosine at positions 31 and 118 for binding to c-CRK-and CRKL-SH2 domains, once the sites have been tyrosine-phosphorylated (7,8). These results suggest that there is overlap and possibly redundancy in the signaling pathway activated by p125 FAK and RAFTK, and additional comparison of these signaling events is warranted.
Tyrosine phosphorylation of paxillin by a protein kinase can be important in interactions between paxillin and other proteins, especially with SH2 domains. Paxillin was originally identified in Rous sarcoma virus-transformed chick embryo fibroblasts through a monoclonal antibody produced by Glenney and Zokas (11). The paxillin cDNA, recently cloned, encodes for a protein with an interesting structure (9,31). Paxillin has four tandem LIM domains C-terminal to various binding sites for SH2 and SH3 domains (9). The interactions between paxillin and SH2-and SH3-containing proteins are important in various signal transduction pathways. Several proteins other than p125 FAK and RAFTK have been shown to tyrosine phosphorylate paxillin, such as v-Crk, v-Src, p210 BCR/ABL , and v-ABL (6,7,10). It has been shown that paxillin binds to the SH2 domains of CRK, CRKL, and Src (7,8,10). Thus, one can envision a role of RAFTK, to promote paxillin binding to other proteins, initially phosphorylating paxillin, and thereafter SH2-domain-containing proteins can bind paxillin.
In summary, we have shown that the tyrosine kinase RAFTK associates with and in vitro phosphorylates paxillin in hematopoietic cells. The effect of RAFTK on paxillin may be important in one of multiple signal transduction pathways. In hematopoietic cells, we show that there is constitutive binding between RAFTK and paxillin in response to IL-3 and the oncogene p210 BCR/ABL . This could be a mechanism through which the cell retains its normal homeostasis.