Cloning and Characterization of Cell Adhesion Kinase , a Novel Protein-tyrosine Kinase of the Focal Adhesion Kinase Subfamily

Department of Biochemistry, Sapporo Medical University School of Medicine, Japan.
Journal of Biological Chemistry (Impact Factor: 4.57). 10/1995; 270(36):21206-19. DOI: 10.1074/jbc.270.36.21206
Source: PubMed


A second protein-tyrosine kinase (PTK) of the focal adhesion kinase (FAK) subfamily, cell adhesion kinase beta (CAK beta), was identified by cDNA cloning. The rat CAK beta is a 115.7-kDa PTK that contains N- and C-terminal domains of 418 and 330 amino acid residues besides the central kinase domain. The rat CAK beta has a homology with mouse FAK over their entire lengths except for the extreme N-terminal 88 residues and shares 45% overall sequence identity (60% identical in the catalytic domain), which indicates that CAK beta is a protein structurally related to but different from FAK. The CAK beta gene is less evenly expressed in a variety of rat organs than the FAK gene. Anti-CAK beta antibody immunoprecipitated a 113-kDa protein from rat brain, 3Y1 fibroblasts, and COS-7 cells transfected with CAK beta cDNA. The tyrosine-phosphorylated state of CAK beta was not reduced on trypsinization, nor enhanced in response to plating 3Y1 cells onto fibronectin. CAK beta localized to sites of cell-to-cell contact in COS-7 transfected with CAK beta cDNA, in which FAK was found at the bottom of the cells. Thus, CAK beta is a PTK possibly participating in the signal transduction regulated by cell-to-cell contacts.

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    • "Many PTKs exhibit key functions in neuronal development and synaptic plasticity. Pyk2, a non-receptor PTK [9], also known as focal adhesion kinase 2 (FAK-2) [10], and calcium-dependent tyrosine kinase (CADTK) [11] (for convenience, hereafter referred to as Pyk2), is a second member of the FAK subfamily and was first reported by Avraham in 1995 [9], [12], [13]. Pyk2 is preferentially expressed in neuronal and hematopoietic cells. "
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    ABSTRACT: Protein tyrosine kinases, which are highly expressed in the central nervous system, are implicated in many neural processes. However, the relationship between protein tyrosine kinases and neurotransmitter release remains unknown. In this study, we found that ionomycin, a Ca2+ ionophore, concurrently induced asynchronous neurotransmitter release and phosphorylation of a non-receptor protein tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2), in clonal rat pheochromocytoma PC12 cells and cerebellar granule cells, whereas introduction of Pyk2 siRNA dramatically suppressed ionomycin-induced neurotransmitter release. Further study indicated that Tyr-402 (Y402) in Pyk2, instead of other tyrosine sites, underwent rapid phosphorylation after ionomycin induction in 1 min to 2 min. We demonstrated that the mutant of Pyk2 Y402 could abolish ionomycin-induced dopamine (DA) release by transfecting cells with recombinant Pyk2 and its mutants (Y402F, Y579F, Y580F, and Y881F). In addition, Src inhibition could prolong phosphorylation of Pyk2 Y402 and increase DA release. These findings suggested that Pyk2 was involved in ionomycin-induced neurotransmitter release through phosphorylation of Y402.
    PLoS ONE 04/2014; 9(4):e94574. DOI:10.1371/journal.pone.0094574 · 3.23 Impact Factor
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    • "Pyk2 has also been shown to localize to macrophage podosomes, where it colocalizes with the αMβ2 integrin, as well as the cytoskeletal-associated proteins paxillin, vinculin and talin, and is phosphorylated upon αMβ2 engagement [14]. Pyk2 and FAK share approximately 45% amino acid identity and 65% similarity [16], [17], [18], [19], [20]. These proteins have similar domain structure that includes an N-terminal FERM (Protein 4.1, Ezrin, Radixin, Moesin) domain, a centrally located kinase domain, two proline-rich regions in the C-terminus, along with a focal adhesion-targeting (FAT) domain [11], [12], [21]. "
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    ABSTRACT: CD45 is a protein tyrosine phosphatase expressed on all cells of hematopoietic origin that is known to regulate Src family kinases. In macrophages, the absence of CD45 has been linked to defects in adhesion, however the molecular mechanisms involved remain poorly defined. In this study, we show that bone marrow derived macrophages from CD45-deficient mice exhibit abnormal cell morphology and defective motility. These defects are accompanied by substantially decreased levels of the cytoskeletal-associated protein paxillin, without affecting the levels of other proteins. Degradation of paxillin in CD45-deficient macrophages is calpain-mediated, as treatment with a calpain inhibitor restores paxillin levels in these cells and enhances cell spreading. Inhibition of the tyrosine kinases proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK), kinases that are capable of mediating tyrosine phosphorylation of paxillin, also restored paxillin levels, indicating a role for these kinases in the CD45-dependent regulation of paxillin. These data demonstrate that CD45 functions to regulate Pyk2/FAK activity, likely through the activity of Src family kinases, which in turn regulates the levels of paxillin to modulate macrophage adhesion and migration.
    PLoS ONE 07/2013; 8(7):e71531. DOI:10.1371/journal.pone.0071531 · 3.23 Impact Factor
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    • "Pyk2 is activated by a number of extracellular signals, including growth factors and ECM adhesion [43] [44]. However, unlike FAK, which is ubiquitously expressed, Pyk2 is restricted to primarily hematopoietic and neuronal tissues, with upregulation in a broader group of tissues only later in development [45] [46], and thus its role in angiogenesis has not been fully characterized. In addition to its compensatory role for FAK in adult angiogenesis, Pyk2 has been shown to be involved in endothelial cell spreading and migration [41], VE-cadherin-mediated cell–cell adhesion [47], and neovessel formation [48] [49]. "
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    ABSTRACT: Angiogenesis is regulated by both soluble growth factors and cellular interactions with the extracellular matrix (ECM). While cell adhesion via integrins has been shown to be required for angiogenesis, the effects of quantitative changes in cell adhesion and spreading against the ECM remain less clear. Here, we show that angiogenic sprouting in natural and engineered three-dimensional matrices exhibited a biphasic response, with peak sprouting when adhesion to the matrix was limited to intermediate levels. Examining changes in global gene expression to determine a genetic basis for this response, we demonstrate a vascular endothelial growth factor (VEGF)-induced upregulation of genes associated with vascular invasion and remodeling when cell adhesion was limited, whereas cells on highly adhesive surfaces upregulated genes associated with proliferation. To explore a mechanistic basis for this effect, we turned to focal adhesion kinase (FAK), a central player in adhesion signaling previously implicated in angiogenesis, and its homologue, proline-rich tyrosine kinase 2 (Pyk2). While FAK signaling had some impact, our results suggested that Pyk2 can regulate both gene expression and endothelial sprouting through its enhanced activation by VEGF in limited adhesion contexts. We also demonstrate decreased sprouting of tissue explants from Pyk2-null mice as compared to wild type mice as further confirmation of the role of Pyk2 in angiogenic sprouting. These results suggest a surprising finding that limited cell adhesion can enhance endothelial responsiveness to VEGF and demonstrate a novel role for Pyk2 in the adhesive regulation of angiogenesis.
    Experimental Cell Research 08/2011; 317(13):1860-71. DOI:10.1016/j.yexcr.2011.05.006 · 3.25 Impact Factor
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