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 (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.

Diffusible signal factor (DSF) family signal-mediated quorum sensing (QS) has been identified in many gram-negative bacteria. This QS pathway of Xanthomonas spp. consists of three major QS components: RpfF, RpfC, and RpfG. The rpfF gene encodes a putative enoyl-CoA hydratase that catalyzes the synthesis of the signal molecule. RpfC and RpfG serve as a two-component system for the perception and transduction of the extracellular DSF family signals. In order to further characterize the QS regulatory network in Xanthomonas citri subsp. citri, we investigated the RpfF, RpfC, and RpfG regulons by using transcriptome analyses. Comparison of the transcriptomes of the QS mutants (rpfF, rpfC, and rpfG) with that of the wild-type strain revealed a core group of genes controlled by all three QS components, suggesting that the RpfC-RpfG two-component system is a major and conserved signal perception and transduction system for DSF family signal-mediated QS in X. citri subsp. citri. The unique genes controlled by RpfF alone indicate the complexity of the QS pathway and the involvement of additional sensory mechanisms in X. citri subsp. citri. The unique genes controlled by RpfC and RpfG, respectively, support the possibility that RpfC and RpfG play broader roles in gene regulation other than transduction of DSF signals.

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The bovine papillomavirus type 1 (BPV-1) E6 oncoprotein can transform fibroblasts and induce anchorage-independent growth and disassembly of the actin stress fibers. We have previously shown that the E6 protein interacts with the focal adhesion protein, paxillin, suggesting a direct role of E6 in the disruption of the actin cytoskeleton. We have now mapped the E6 binding sites on paxillin to the LD motif repeats region, which has been implicated in mediating paxillin binding to two other focal adhesion proteins, vinculin and the focal adhesion kinase. The five LD motif repeats identified in paxillin do not contribute equally to its interaction with E6. The first LD repeat is most critical for paxillin binding to E6 both in vitro and in vivo. Furthermore, the binding of recombinant wild-type E6 protein to paxillin blocked the interaction of several cellular proteins with paxillin, including vinculin and the focal adhesion kinase. A mutant E6 protein (H105) which does not bind to paxillin had no effect on the binding of these cellular proteins to paxillin. These data suggest that E6 disruption of the actin stress fibers occurs through blocking the interaction of paxillin with its cellular effectors such as vinculin and the focal adhesion kinase.Cellular adhesion to the extracellular matrix involves signals that are essential in many processes including cell morphology, cell division, cell motility, and tumor metastasis (for review, see Refs. 1-4). Focal adhesions are specialized structures where cells adhere to the extracellular matrix through a network of actin cytoskeleton (for review, see Refs. 5 and 6). Focal adhesions contain integrins, tensin, vinculin, the focal adhesion kinase (FAK), 1 and paxillin. Paxillin is tyrosine phosphorylated in response to a variety of stimuli including cell adhesion, alterations in the actin cytoskeleton, and treatment with growth factors (7). Tyrosine phosphorylation of focal adhesion proteins is closely associated with changes in the structure of the actin cytoskeleton, although the precise downstream molecular consequences of such phosphorylation are currently unknown. Several lines of evidence have suggested that FAK is, at least in part, responsible for the tyrosine phosphorylation of paxillin observed in vivo (8, 9). Paxillin also binds to vinculin (10), an abundant cytoskeletal protein that is important in the assembly of the actin cytoskeleton, possibly through its interactions with other focal adhesion proteins, ␣-actinin, and actin (for review, see Ref. 6). The importance of a role for paxillin in cell signaling is suggested by the fact that paxillin is the target of several viral oncoproteins including v-Src (11), v-Crk (12), v-Abl (13), and the E6 oncoprotein of bovine papillomavirus type-1 (BPV-1) from our previous work (14). The interaction of paxillin with BPV-1 E6 is specific for transformation competent E6 proteins, and the expression of E6 results in the disruption of the actin stress fibers (14), suggesting that the binding of BPV-1 E6 to paxillin may be impor...

Phosphoinositide-dependent kinase-1 (PDK-1) is a serine/threonine protein kinase that phosphorylates members of the conserved AGC kinase superfamily, including AKT and PKC, and is implicated in important cellular processes including survival, metabolism and tumorigenesis. In large cohorts of nevi and melanoma samples, PDK1 expression was significantly higher in primary melanoma, compared with nevi, and was further increased in metastatic melanoma. PDK1 expression suffices for its activity, due to auto-activation, or elevated phosphorylation by phosphoinositide 3'-OH-kinase (PI 3-K). Selective inactivation of Pdk1 in the melanocytes of BrafV600E::Pten−/− or BrafV600E::Cdkn2a−/−::Pten−/− mice delayed the development of pigmented lesions and melanoma induced by systemic or local administration of 4-HT. Melanoma invasion and metastasis were significantly reduced or completely prevented by Pdk1 deletion. Administration of the PDK1 inhibitor GSK2334470 (PDKi) effectively delayed melanomagenesis and metastasis in BrafV600E::Pten−/− mice. Pdk1−/− melanomas exhibit a marked decrease in the activity of AKT, P70S6K and PKC. Notably, PDKi was as effective in inhibiting AGC kinases and colony forming efficiency of melanoma with Pten WT genotypes. Gene expression analyses identified Pdk1-dependent changes in FOXO3a-regulated genes and inhibition of FOXO3a restored proliferation and colony formation of Pdk1−/− melanoma cells. Our studies provide direct genetic evidence for the importance of PDK1, in part through FOXO3a-dependent pathway, in melanoma development and progression.

LKB1 is a tumor susceptibility gene for the Peutz-Jeghers cancer syndrome and is a target for mutational inactivation in sporadic human malignancies. LKB1 encodes a serine/threonine kinase that has critical roles in cell growth, polarity and metabolism. A novel and important function of LKB1 is its ability to regulate the phosphorylation of CREB-regulated transcription co-activators (CRTCs) whose aberrant activation is linked with oncogenic activities. However, the roles and mechanisms of LKB1 and CRTC in the pathogenesis of esophageal cancer have not been previously investigated. In this study, we observed altered LKB1-CRTC signaling in a subset of human esophageal cancer cell lines and patient samples. LKB1 negatively regulates esophageal cancer cell migration and invasion in vitro. Mechanistically, we determined that CRTC signaling becomes activated because of LKB1 loss, which results in the transcriptional activation of specific downstream targets including LYPD3, a critical mediator for LKB1 loss-of-function. Our data indicate that de-regulated LKB1-CRTC signaling might represent a crucial mechanism for esophageal cancer progression.