Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Imatinib is an inhibitor of the Abl tyrosine kinase domain that is effective in the treatment of chronic myelogenic leukemia. Although imatinib binds tightly to the Abl kinase domain, its affinity for the closely related kinase domain of c-Src is at least 2,000-fold lower. Imatinib recognition requires a specific inactive conformation of the kinase domain, in which a conserved Asp-Phe-Gly (DFG) motif is flipped with respect to the active conformation. The inability of c-Src to readily adopt this flipped DFG conformation was thought to underlie the selectivity of imatinib for Abl over c-Src. Here, we present a series of inhibitors (DSA compounds) that are based on the core scaffold of imatinib but which bind with equally high potency to c-Src and Abl. The DSA compounds bind to c-Src in the DFG-flipped conformation, as confirmed by crystal structures and kinetic analysis. The origin of the high affinity of these compounds for c-Src is suggested by the fact that they also inhibit clinically relevant Abl variants bearing mutations in a structural element, the P-loop, that normally interacts with the phosphate groups of ATP but is folded over a substructure of imatinib in Abl. Importantly, several of the DSA compounds block the growth of Ba/F3 cells harboring imatinib-resistant BCR-ABL mutants, including the Thr315Ile "gatekeeper" mutation, but do not suppress the growth of parental Ba/F3 cells.
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PMID:Equally potent inhibition of c-Src and Abl by compounds that recognize inactive kinase conformations. 1927 51

Adipocyte fatty acid-binding protein (AFABP/aP2) facilitates the intracellular solubilization and trafficking of lipids within the aqueous environment of the cell. Studies in the AFABP/aP2 knock-out mouse suggest that the protein may have roles in cellular processes broader than lipid transport. We present herein the finding that AFABP/aP2 interacts with JAK2 in a fatty acid-dependent manner. This interaction was established using yeast two-hybrid analysis, co-immunoprecipitation from adipose tissue, and 3T3-L1 adipocytes as well as in 293 cells overexpressing JAK2 and AFABP/aP2. Mutational analysis of AFABP/aP2 (R126L/Y128F) revealed that fatty acid binding activity is necessary for the interaction and that Asp(18) of the helix-turn-helix motif forms a component of the interaction domain. Mutational analysis of JAK2 (Y1007F/Y1008F) revealed that AFABP/aP2 associates with the basal unphosphorylated form of the protein. Interleukin-6, but not interleukin-10, stimulated phosphorylation of STAT3, and induction of SOCS3 mRNA expression were potentiated in a time- and dose-dependent manner in macrophage cell lines derived from AFABP/aP2-EFABP/mal1 double knock-out mice relative to cells from wild type animals. These results suggest that ligand-bound AFABP/aP2 binds to and attenuates JAK2 signaling and establishes a new role for AFABP/aP2 as a fatty acid sensor affecting cellular metabolism via protein-protein interactions.
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PMID:Interaction of adipocyte fatty acid-binding protein (AFABP) and JAK2: AFABP/aP2 as a regulator of JAK2 signaling. 1931 53

Src family kinases (SFKs) are commonly deregulated in cancer cells. Among other functions, SFKs are critical for cellular migration and invasion. SFK inhibitors are being studied as targeted cancer drugs, but there are no biomarkers for noninvasive assessment of SFK inhibition. The aim of this study was to evaluate whether imaging of alpha(V)beta(3) integrin activity with positron emission tomography (PET) and [(64)Cu]DOTA-cyclo-(Arg-Gly-Asp-dPhe-Lys) {[(64)Cu]DOTA-c(RGDfK)} can be used for monitoring response to the SFK inhibitor dasatinib. Severe combined immunodeficient mice bearing U87MG xenografts were gavaged daily over 72 hours with 72 or 95 mg/kg of dasatinib or vehicle. Tumor uptake of [(64)Cu]DOTA-c(RGDfK) was measured by small-animal PET. In parallel, fluorodeoxyglucose (FDG) scans were performed to assess tumor metabolism in response to dasatinib treatment. Dasatinib significantly (P<0.0001) reduced [(64)Cu]DOTA-c(RGDfK) uptake by up to 59% in U87MG xenografts [2.10+/-0.14% injected dose/gram (ID/g) in the 95 mg/kg group and 3.12+/-0.18% ID/g in the 72 mg/kg group, versus 5.08+/-0.80% ID/g in controls]. In contrast, tumor FDG uptake showed no significant reduction with dasatinib therapy (8.13+/-0.45% ID/g in treated versus 10.39+/-1.04% ID/g in controls; P=0.170). Histologically, tumors were viable at the time of the follow-up PET scan but showed inhibition of focal adhesion kinase. Continued dasatinib treatment resulted in a significant inhibition of tumor growth (tumor size on day 10 of therapy: 21.13+/-2.60 mm(2) in treated animals versus 122.50+/-17.68 mm(2) in controls; P=0.001). [(64)Cu]DOTA-c(RGDfK) may provide a sensitive means of monitoring tumor response to SFK inhibition in alpha(V)beta(3)-expressing cancers early in the course of therapy.
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PMID:Noninvasive imaging of alphaVbeta3 function as a predictor of the antimigratory and antiproliferative effects of dasatinib. 1931 69

The adhesion and proliferation of human adipo-stromal cells was investigated for poly(ethylene terephthalete) (PET) films of two-dimensional (2D) substrate and non-woven fabrics of three-dimensional (3D) substrate after seeding at the same cell density and culturing at the same medium volume to surface area of the substrates ratio. When seeded by a static seeding method, more cells adhered on the film than on the non-woven fabric. However, the number ratio of cells proliferated to those initially adhered was similar. For the non-woven fabric, cell proliferation was enhanced by an agitated culture method. NaOH treatment introduced carboxyl groups into the surface of substrates, irrespective of the substrate type. Cell adhesion of a peptide (Arg-Gly-Asp, RGD) was chemically immobilized through the carboxyl groups on the non-woven fabric surface at a density of 10 pmol/cm2. RGD immobilization significantly increased the number of cells adhered after the agitated seeding method, but did not affect the cell proliferation. Phosphorylation of focal adhesion kinase (FAK) was also enhanced by the RGD immobilization on the PET non-woven fabrics.
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PMID:Adhesion and proliferation of human adipo-stromal cells for two- or three-dimensional poly(ethylene terephthalate) substrates with or without RGD immobilization. 1932 86

We previously established a simple method to immobilize the Arg-Gly-Asp (RGD) peptide on polycaprolactone (PCL) two-dimensional film surfaces that significantly improved bone marrow stromal cell (BMSC) adhesion to these films. The current work extends this modification strategy to three-dimensional (3D) PCL scaffolds to investigate BMSC attachment, cellular distribution and cellularity, signal transduction and survival on the modified PCL scaffold compared to those on the untreated ones. The results demonstrated that treatment of 3D PCL scaffold surfaces with 1,6-hexanediamine introduced the amino functional groups onto the porous PCL scaffold homogenously as detected by a ninhydrin staining method. Followed by the cross-linking reaction, RGDC peptide was successfully immobilized on the surface of PCL scaffold. Although the static seeding method used in this study caused heterogeneous cell distribution, the RGD-modified PCL scaffold still demonstrated the improved BMSC attachment and cellular distribution in the scaffold. More importantly, the integrin-mediated signal transduction FAK-PI3K-Akt pathway was significantly up-regulated by RGD modification and a subsequent increase in cell survival and growth was found in the modified scaffold. The present study introduces an easy method to immobilize RGD peptide on the 3D porous PCL scaffold and provides further evidence that modification of 3D PCL scaffolds with RGD peptides elicits specific cellular responses and improves the final cell-biomaterial interaction.
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PMID:The interaction between bone marrow stromal cells and RGD-modified three-dimensional porous polycaprolactone scaffolds. 1948 19

JAK2 (Janus kinase 2) is essential for cytokine receptor signaling, and several lines of evidence support a causal role of an activating JAK2 mutation in myeloproliferative disorders. JAK2 activity is autoinhibited by its pseudokinase domain in the basal state, and the inhibition is released by cytokine stimulation; how engagement of the cognate receptor triggers this release is unknown. From a functional screen for gain-of-function JAK2 mutations, we discovered 13 missense mutations, nine in the pseudokinase domain and four in the Src homology 2 (SH2)-pseudokinase domain linker. These mutations identified determinants for autoinhibition and inducible activation in JAK2. Two of the mutants, K539I and N622I, resulted in erythrocytosis in mice. Scanning mutagenesis of the SH2-pseudokinase domain linker indicated that its N-terminal part was essential for interaction of JAK2 with the Epo receptor, whereas certain mutations in the C-terminal region conferred constitutive activation. We further showed that substitutions for Glu(543)-Asp(544) in this linker or Leu(611), Arg(683), or Phe(694) in the hinge proximal region of the pseudokinase domain resulted in activated JAK2 mutants that could not be further stimulated by Epo. These results suggest that the SH2-pseudokinase domain linker acts as a switch that relays cytokine engagement to JAK2 activation by flexing the pseudokinase domain hinge.
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PMID:A JAK2 interdomain linker relays Epo receptor engagement signals to kinase activation. 1963 29

Cell cycle regulation by differentiation signals is critical for eukaryote development. We investigated the roles of bone morphogenetic protein (BMP)-4, an important stimulator of osteoblast differentiation and bone formation, in regulating cell cycle distribution in four osteoblast-like cell lines and mouse primary osteoblasts, and the underlying mechanisms. In all cells used, BMP-4 induced G(0)/G(1) arrest. The molecular basis of the BMP-4 effect was analyzed, and the presentation on molecular mechanism is focused on human MG63 cells. BMP-4 induced p21(CIP1) and p27(KIP1) expressions and hence cell differentiation but had no effects on the expressions of cyclins A, B1, D1, and E, cyclin-dependent protein kinase-2, -4, and -6. Using specific small interfering RNA (siRNA), we found that BMP-4-induced G(0)/G(1) arrest, and p21(CIP1) and p27(KIP1) expressions were mediated by BMP receptor type IA (BMPRIA)-specific Sma- and Mad-related protein (Smad)1/5. BMP-4 induced transient phosphorylations of ERK; transfection of MG63 cells with ERK2, but not ERK1, -specific siRNA inhibited the BMP-4-induced responses in MG63 cells. Pretreatment of MG63 cells with Arg-Gly-Asp-Ser, which blocks the cell-extracellular matrix interaction, or transfection with beta(3) integrin-specific siRNA inhibited BMP-4-induced ERK and Smad1/5 phosphorylations. BMP-4 induced transient increases in associations of beta(3)-integrin with focal adhesion kinase and Shc, the dominant-negative mutants of which inhibited BMP-4-induced ERK and Smad1/5 phosphorylations. Our results indicate that BMP-4 induces G(0)/G(1) arrest and hence differentiation in osteoblast-like cells through increased expressions of p21(CIP1) and p27(KIP1), which are mediated by BMPRIA-specific Smad1/5. The extracellular matrix/beta(3) integrin/ focal adhesion kinase/Shc/ERK2 signaling pathway is involved in these BMP-4-induced responses in osteoblast-like cells.
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PMID:BMP-4 induction of arrest and differentiation of osteoblast-like cells via p21 CIP1 and p27 KIP1 regulation. 1981 88

Insulin stimulates endothelial NO (nitric oxide) synthesis via PKB (protein kinase B)/Akt-mediated phosphorylation and activation of eNOS (endothelial NO synthase) at Ser-1177. In previous studies, we have demonstrated that stimulation of eNOS phosphorylation at Ser-1177 may be required, yet is not sufficient for insulin-stimulated NO synthesis. We therefore investigated the role of phosphorylation of eNOS at alternative sites to Ser-1177 as candidate parallel mechanisms contributing to insulin-stimulated NO synthesis. Stimulation of human aortic endothelial cells with insulin rapidly stimulated phosphorylation of both Ser-615 and Ser-1177 on eNOS, whereas phosphorylation of Ser-114, Thr-495 and Ser-633 was unaffected. Insulin-stimulated Ser-615 phosphorylation was abrogated by incubation with the PI3K (phosphoinositide 3-kinase) inhibitor wortmannin, infection with adenoviruses expressing a dominant-negative mutant PKB/Akt or pre-incubation with TNFalpha (tumour necrosis factor alpha), but was unaffected by high culture glucose concentrations. Mutation of Ser-615 to alanine reduced insulin-stimulated NO synthesis, whereas mutation of Ser-615 to aspartic acid increased NO production by NOS in which Ser-1177 had been mutated to an aspartic acid residue. We propose that the rapid PKB-mediated stimulation of phosphorylation of Ser-615 contributes to insulin-stimulated NO synthesis.
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PMID:Insulin-stimulated phosphorylation of endothelial nitric oxide synthase at serine-615 contributes to nitric oxide synthesis. 1992 57

Denaturation of extracellular matrix proteins exposes cryptic binding sites. It is hypothesized that binding of cell adhesion receptors to these cryptic binding sites regulates cellular behaviour during tissue repair and regeneration. To test this hypothesis, we quantify the adhesion of pre-osteoblastic cells to native (Col) and partially-denatured (pdCol) collagen I using single-cell force spectroscopy. During early stages of cell attachment (< or =180 s) pre-osteoblasts (MC3T3-E1) adhered significantly stronger to pdCol compared to Col. RGD (Arg-Gly-Asp)-containing peptides suppressed this elevated cell adhesion. We show that the RGD-binding alpha(5)beta(1)- and alpha(v)-integrins mediated pre-osteoblast adhesion to pdCol, but not to Col. On pdCol pre-osteoblasts had a higher focal adhesion kinase tyrosine-phosphorylation level that correlated with enhanced spreading and motility. Moreover, pre-osteoblasts cultured on pdCol showed a pronounced matrix mineralization activity. Our data suggest that partially-denatured collagen exposes RGD-motifs that trigger binding of alpha(5)beta(1)- and alpha(v)-integrins. These integrins initiate cellular processes that stimulate osteoblast adhesion, spreading, motility and differentiation. Taken together, these quantitative insights reveal an approach for the development of alternative collagen I- based surfaces for tissue engineering applications.
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PMID:The effect of unlocking RGD-motifs in collagen I on pre-osteoblast adhesion and differentiation. 2005 43

The extracellular matrix (ECM) is thought to be an essential component of tissue scaffolding and engineering because it fulfills fundamental functions related to cell adhesion, migration, and three-dimensional organization. Natural ECM preparations, however, are challenging to work with because they are comprised of macromolecules that are large and insoluble in their functional state. Functional fragments of ECM macromolecules are a viable answer to this challenge, as demonstrated by the RGD-based engineered scaffolds, where the tri-peptide, Arg-Gly-Asp (RGD), represents the minimal functional unit of fibronectin and related ECM. Laminins (Ln) are main components of epithelial tissues, since they enter into the composition of basement membranes. Application of Ln to epithelial tissue engineering would be desirable, since they could help mimic ideal functional conditions for both lining and glandular epithelial tissues. However, functional fragments of Ln that could be used in artificial settings have not been characterized in detail. In this paper, we describe the production and application of the recombinant LG4 (rLG4) fragment of laminin-332 (Ln-332), and show that it mimics three fundamental functional properties of Ln-332: integrin-mediated cell adhesion, spreading, and migration. Adhesive structures formed by cells on rLG4 closely resemble those formed on Ln-332, as judged by microscopy-based analyses of their molecular composition. As on Ln-332, focal adhesion kinase (FAK) is phosphorylated in cells adhering to rLG4, and colocalized with other focal adhesion components. We conclude that rLG4 could be a useful substitute to recapitulate, in vitro, the tissue scaffolding properties of Ln-332.
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PMID:The role of a recombinant fragment of laminin-332 in integrin alpha3beta1-dependent cell binding, spreading and migration. 2034 31


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