The Related Adhesion Focal Tyrosine Kinase Forms a Complex with Paxillin in Hematopoietic Cells

Abstract

Related adhesion focal tyrosine kinase (RAFTK), also known as proline-rich tyrosine kinase 2 and cellular adhesion kinase ␤, 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 p125 FAK 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 p125 FAK , 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 p125 FAK , may be important in phosphotyrosine-signaling events within the focal adhesion. Related adhesion focal tyrosine kinase (RAFTK)1 is a protein tyrosine kinase that is a member of the focal adhesion kinase (FAK) subfamily (1-4). RAFTK was originally cloned from a cDNA library from CMK megakaryocytic cells (1). The cDNA encoding RAFTK is 3.6 kilobases with a translated product of 123 kDa. RAFTK has a "focal adhesion-targeting" domain, which is 52% homologous to the focal adhesion-targeting domain of p125 FAK , both of which are located in the C terminus of their respective proteins. RAFTK lacks a transmembrane region, myristylation sites, and SH2 and SH3 domains. These protein tyrosine kinase have highly related kinase domains (amino acids 419 -680 in human RAFTK and amino acids 390 -650 in human p125 FAK ), but their N-and C-terminal domains differ (1).Recently, RAFTK has been suggested to play a role in signal 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...

Expression of the focal adhesion protein paxillin in lung cancer and its relation to cell motility

“…We have performed cell staining for paxillin and vinculin in the human diploid lung line MRC-5 and various NSCLC cell lines (Calu-1 and SK-LU-1) (Figure 1). Using the anti-paxillin antibody 5H11, paxillin is localized to the focal adhesion in these cells (Salgia et al, 1996a). As a control, vinculin is in the focal adhesions as expected.…”

“…b1 and b2 integrin activation on the cell surface also lead to phosphorylation of paxillin (Charlesworth et al, 1996). Multiple kinases, including p125 FAK and RAFTK, can directly interact with paxillin (Salgia et al, 1996a).…”

PMA-induced phenotypic changes in K562 cells: MAPK-dependent and -independent events

“…57 We also demonstrated that PMA treatment induces the expression of tyrosine phosphorylated Pyk2 (Figures 3 and 4), an event thought to be involved in the phosphorylation of paxillin. 58 Given the fact that paxillin phosphorylation was seen only under conditions that lead to differentiation, we favor the hypothesis that it becomes phosphorylated due to the enhanced expression of integrin receptors associated with PMA-induced differentiation. 21 Phosphorylated Pyk2 forms a complex with CrkL in differentiated cells (Figure 4).…”

RAFTK/Pyk2 tyrosine kinase mediates the association of p190 RhoGAP with RasGAP and is involved in breast cancer cell invasion

“…Association of RAFTK with di erent protein tyrosine kinases (PTKs) such as Src, Fyn, and Csk as well as with the adaptor protein Grb2 was reported (Dikic et al, 1996;Li et al, 1996). RAFTK was shown to interact with several focal adhesion molecules such as paxillin, p130cas and HAF1 (Astier et al, 1997a,b;Hiregowdara et al, 1997;Salgia et al, 1996). In this report, we have investigated the role of RAFTK in HRG-mediated signaling in breast cancer cells.…”