Paxillin is a 68-kDa focal adhesion protein that is phosphorylated on tyrosine residues in fibroblasts in response to transformation by v-src, treatment with platelet-derived growth factor, or cross-linking of integrins. Paxillin has been shown to have binding sites for the SH3 domain of Src and the SH2 domain of Crk in vitro and to coprecipitate with two other focal adhesion proteins, vinculin and focal adhesion kinase (p125fak). After preliminary studies showed that paxillin was a substrate for the hematopoietic oncogene p210BCR/ABL, we investigated the role of this protein in hematopoietic cell transformation and signal transduction. A full-length length cDNA encoding human paxillin was cloned, revealing multiple protein domains, including four tandem LIM domains, a proline-rich domain containing a consensus SH3 binding site, and three potential Crk-SH2 binding sites. The paxillin gene was localized to chromosome 12q24 by fluorescence in situ hybridization analysis. A chicken paxillin cDNA was also cloned and is predicted to encode a protein approximately 90% identical to human paxil-lin. Paxillin coprecipitated with p210BCR/ABL and multiple other cellular proteins in myeloid cell lines, suggesting the formation of multimeric complexes. In normal hematopoietic cells and myeloid cell lines, tyrosine phosphorylation of paxillin and coprecipitation with other cellular proteins was rapidly and transiently induced by interleukin-3 and several other hematopoietic growth factors. The predicted structure of paxillin implicates this molecule in protein-protein interactions involved in signal transduction from growth factor receptors and the BCR/ABL oncogene fusion protein to the cytoskeleton.

The BCR/ABL oncogene causes human chronic myelogenous leukemia (CML), a myeloproliferative disease characterized by massive expansion of hematopoietic progenitor cells and cells of the granulocyte lineage. When transfected into murine hematopoietic cell lines, BCR/ABL causes cytokine-independence and enhances viability. There is also growing evidence that p210 BCR/ABL affects cytoskeletal structure. p210 BCR/ABL binds to actin, and several cytoskeletal proteins are tyrosine phosphorylated by this oncoprotein. Also, at least one aspect of cytoskeletal function is abnormal, in that the affinity of ␤ 1 integrins for fibronectin is altered in CML cells. However, isolated changes in ␤ 1 integrin function would be unlikely to explain the clinical phenotype of CML. We used time-lapse video microscopy to study cell motility and cell morphology on extracellular cell matrix protein-coated surfaces of a series of cell lines before and after transformation by BCR/ABL . BCR/ABL was associated with a striking increase in spontaneous motility, membrane ruffling, formation of long actin extensions (filopodia) and accelerated the rate of protrusion and retraction of pseudopodia on fibronectin-coated surfaces. Also, while untransformed cells were sessile for long periods, BCR/ABLtransformed cells exhibited persistent motility, except for brief periods during cell division. Using cell lines transformed by a temperature-sensitive mutant of BCR/ABL, these kinetic abnormalities of cytoskeletal function were shown to require BCR/ABL tyrosine kinase activity. Similar abnormalities of cytoskeletal function on fibronectin-coated surfaces were observed when hematopoietic progenitor cells purified by CD34 selection from patients with CML were compared with CD34 positive cells from normal individuals. Interestingly, ␣ -interferon treatment was found to slowly revert the abnormal motility phenotype of BCR/ABL -transformed cells towards normal. The increase in spontaneous motility and other defects of cytoskeletal function described here will be useful biological markers of the functional effects of BCR/ABL in hematopoietic cells. ( J. Clin. Invest.

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...

The Philadelphia chromosome translocation generates a chimeric oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML). In primary neutrophils from patients with CML, the major novel tyrosine-phosphorylated protein is CRKL, an SH2-SH3-SH3 linker protein which has an overall homology of 60% to CRK, the human homologue of the v-crk oncogene product. Anti-CRKL immunoprecipitates from CML cells, but not normal cells, were found to contain p210BCR/ABL and c-ABL. Several other phosphoproteins were also detected in anti-CRKL immunoprecipitates, one of which has been identified as paxillin, a 68-kDa focal adhesion protein which we have previously shown to be phosphorylated by p210BCR/ABL. Using GST-CRKL fusion proteins, the SH3 domains of CRKL were found to bind c-ABL and p210BCR/ABL, while the SH2 domain of CRKL bound to paxillin, suggesting that CRKL could physically link p210BCR/ABL to paxillin. Paxillin contains three tyrosines in Tyr-X-X-Pro (Y-X-X-P) motifs consistent with amino acid sequences predicted to be optimal for binding to the CRKL-SH2 domain (at positions Tyr-31, Tyr-118, and Tyr-181). Each of these tyrosine residues was mutated to a phenylalanine residue, and in vitro binding assays indicated that paxillin tyrosines at positions 31 and 118, but not 181, are likely to be involved in CRKL-SH2 binding. These results suggest that the p210BCR/ABL oncogene may be physically linked to the focal adhesion-associated protein paxillin in hematopoietic cells by CRKL. This interaction could contribute to the known adhesive defects of CML cells.

The Philadelphia chromosome (Ph) translocation generates a chimeric tyrosine kinase oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML) and a type of acute lymphoblastic leukemia (ALL). In primary samples from virtually all patients with CML or Ph ؉ ALL, the CRKL adapter protein is tyrosine phosphorylated and physically associated with p210 BCR/ABL . CRKL has one SH2 domain and two SH3 domains and is structurally related to c-CRK-II (CRK) and the v-Crk oncoprotein. We have previously shown that CRKL, but not the related adapter protein c-CRK, is tyrosine phosphorylated in cell lines transformed by BCR/ABL, and that CRKL binds to BCR/ABL through the CRKL-SH3 domains. Furthermore, the CRKL-SH2 domain has been shown to bind one or more cellular proteins, one of which is p120CBL . Here we demonstrate that another cellular protein linked to BCR/ABL through the CRKL-SH2 domain is p130 CAS . p130 CAS was found to be tyrosine phosphorylated and associated with CRKL in BCR/ABL expressing cell lines and in samples obtained from CML and ALL patients, but not in samples from controls. In both normal and BCR/ABL transformed cells, p130 CAS was detected in focal adhesion-like structures, as was BCR/ABL. In normal cells, the focal adhesion proteins tensin, p125 FAK , and paxillin constitutively associated with p130 CAS . However, in BCR/ABL transformed cells, the interaction between p130 CAS and tensin was disrupted, while the associations between p130 CAS , p125 FAK , and paxillin were unaffected. These results suggest that the BCR/ABL oncogene could alter the function of p130 CAS in at least three ways: tyrosine phosphorylation, inducing constitutive binding of CRKL to a domain in p130 CAS containing Tyr-X-X-Pro motifs (substrate domain), and disrupting the normal interaction of p130 CAS with the focal adhesion protein tensin. These alterations in the structure of signaling proteins in focal adhesion like structures could contribute to the known adhesion abnormalities in CML cells.

PI3K was investigated. Far Western blotting with a CRKL-SH3 glutathione S-transferase fusion protein showed that CRKL binds directly to p85 PI3K in vitro. However, although a small amount of CRKL was preassociated with p85 PI3K , the interaction was increased after SF stimulation, suggesting that the interactions of these three proteins are complex. We conclude that SF induces the formation of a signaling complex potentially containing CRKL and p120 CBL , both of which bind to c-Kit through p85 PI3K. These data suggest that one function of CRKL in normal cells might be to recruit signaling molecules such as CBL into a complex with PI3K. Such complexes could be important in propagating signals involving PI3K such as gene expression and adhesion.

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Metastatic carcinoma of unknown primary is a common problem, accounting for up to 10-15% of all solid tumours at presentation. Proper identification of the site of origin has prognostic and therapeutic significance. Prior immunohistochemical methods to identify the site of origin have been useful in a limited number of cases. Differential cytokeratin staining may be useful in this setting, particularly in identifying metastases from lung cancer. We have identified 144 cases of metastatic carcinoma of unknown primary to bone, lung or liver at Brigham and Women's Hospital between 1 January 1997 and 1 July 1998. Cytokeratin (CK) 7 and CK20 were used in 75 of these cases to narrow down the possible sites of the primary tumours. All of these cases were ambiguous as to the site of the primary tumour. Forty-five cases were CK7+/CK20-, 15 cases were CK7-/CK20-, 9 cases were CK7-/CK20+ and 6 cases were CK7+/CK20+. Three of the cases were selected for detailed presentation and discussion as well as a discussion of the pertinent literature. Overall, the CK7+/CK20- phenotype favours a lung primary, the CK7+/CK20+ phenotype strongly favours transitional cells (urothelial) carcinoma, the CK7-/CK20+ phenotype favours colorectal carcinoma, while the CK7-/CK20- profile is not helpful.