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)

Src homology region 2 (SH2) domains are present in many proteins involved in signal transduction. In nonreceptor protein tyrosine kinases the SH2 domain has been implicated in regulation of tyrosine kinase activity and in mediating interactions involved in downstream signaling. Different SH2 domains exhibit distinct binding specificities for both phosphotyrosine- and phosphoserine/phosphothreonine-containing proteins. We show that different SH2 domains are not functionally equivalent within the context of the c-ABL1b protooncogene. c-ABL1b, altered by replacement of its SH2 domain with the N-terminal SH2 domain of Ras GTPase-activating protein, exhibited activated transforming capability, caused intracellular tyrosine phosphorylation of p62, and was relocalized from nucleus to cytoplasm. This en bloc substitution apparently uncouples two distinct functions of the SH2 domain so that c-ABL escapes normal regulatory control while it retains the capability to elicit signals that promote transformation. The SH2 domain of the ARG protein tyrosine kinase, which shares high amino acid-sequence homology with the SH2 domain of ABL, was less effective in activating the oncogenic potential of c-ABL. The effects that the N-terminal SH2 domain of Ras GTPase-activating protein has in the context of c-ABL resemble the effects of deleting the SH3 domain. Thus, the SH2 and SH3 domains may have coordinate roles as regulatory control elements within the context of c-ABL.
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PMID:En bloc substitution of the Src homology region 2 domain activates the transforming potential of the c-Abl protein tyrosine kinase. 768 3

Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L. ) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [gamma-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60(src) (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species.
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PMID:Protein phosphorylation during coconut zygotic embryo development 973 45

We have investigated the interaction between Cbl and the Src-related tyrosine kinase Fyn. Fyn was observed to be constitutively associated with Cbl in lysates of several different cell types including the interleukin-3-dependent murine myeloid cell line 32Dcl3, and the prolactin-dependent rat thymoma cell line Nb2. Binding studies indicated that Cbl could bind to glutathione S-transferase (GST) fusion proteins encoding the unique, Src homology domain 3 (SH3), and SH2 domains of Fyn, Hck, or Lyn. Fusion proteins encoding either the SH3 or SH2 domains of Fyn bound to Cbl as effectively as the fusion protein encoding the unique, SH3, and SH2 domains of Fyn. The Fyn SH2 domain bound to both tyrosine-phosphorylated and nonphosphorylated Cbl, implying that this interaction might be phosphotyrosine-independent. Binding of the Fyn SH2 domain to Cbl was not disrupted by the addition of phosphotyrosine, phosphoserine, or phosphothreonine. A GST fusion protein encoding the proline-rich region of Cbl bound to Fyn present in a total cell lysate. Far Western blot analysis also indicated that the SH3 domain of Fyn bound preferentially to the proline-rich region of Cbl. The addition of [gamma-32P]ATP to either anti-Cbl immunoprecipitates or anti-Fyn immunoprecipitates resulted in the phosphorylation of both Cbl and Fyn as demonstrated by immunoprecipitation of the phosphorylated proteins with specific antisera. Fyn directly phosphorylated a GST fusion protein containing the C-terminal region of Cbl (GST-CBL-LZIP). In contrast, immunoprecipitated JAK2 was not able to phosphorylate this same region of Cbl. The GST-CBL-LZIP fusion protein contains a binding site for the SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase, which mapped to Tyr731, which is present in the sequence YEAM. Mutation of Tyr731 in GST-CBL-LZIP eliminated binding of the p85 subunit of phosphatidylinositol 3-kinase and substantially reduced the phosphorylation of this fusion protein by Fyn, despite the presence of four other tyrosine residues in this fusion protein. These data are consistent with the hypothesis that Cbl represents a substrate for Src-like kinases that are activated in response to the engagement of cell surface receptors, and that Src-like kinases are responsible for the phosphorylation of a tyrosine residue in Cbl that may regulate activation of phosphatidylinositol 3-kinase.
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PMID:Fyn associates with Cbl and phosphorylates tyrosine 731 in Cbl, a binding site for phosphatidylinositol 3-kinase. 989 Sep 70

Protein-interacting modules help determine the specificity of signal transduction events, and protein phosphorylation can modulate the assembly of such modules into specific signaling complexes. Although phosphotyrosine-binding modules have been well-characterized, phosphoserine- or phosphothreonine-binding modules have not been described. WW domains are small protein modules found in various proteins that participate in cell signaling or regulation. WW domains of the essential mitotic prolyl isomerase Pin1 and the ubiquitin ligase Nedd4 bound to phosphoproteins, including physiological substrates of enzymes, in a phosphorylation-dependent manner. The Pin1 WW domain functioned as a phosphoserine- or phosphothreonine-binding module, with properties similar to those of SRC homology 2 domains. Phosphoserine- or phosphothreonine-binding activity was required for Pin1 to interact with its substrates in vitro and to perform its essential function in vivo.
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PMID:Function of WW domains as phosphoserine- or phosphothreonine-binding modules. 1008 27

Endothelial cells release nitric oxide (NO) acutely in response to increased laminar fluid shear stress, and the increase is correlated with enhanced phosphorylation of endothelial nitric-oxide synthase (eNOS). Phosphoamino acid analysis of eNOS from bovine aortic endothelial cells labeled with [(32)P]orthophosphate demonstrated that only phosphoserine was present in eNOS under both static and flow conditions. Fluid shear stress induced phosphate incorporation into two specific eNOS tryptic peptides as early as 30 s after initiation of flow. The flow-induced tryptic phosphopeptides were enriched, separated by capillary electrophoresis with intermittent voltage drops, also known as "peak parking," and analyzed by collision-induced dissociation in a tandem mass spectrometer. Two phosphopeptide sequences determined by tandem mass spectrometry, TQpSFSLQER and KLQTRPpSPGPPPAEQLLSQAR, were confirmed as the two flow-dependent phosphopeptides by co-migration with synthetic phosphopeptides. Because the sequence (RIR)TQpSFSLQER contains a consensus substrate site for protein kinase B (PKB or Akt), we demonstrated that LY294002, an inhibitor of the upstream activator of PKB, phosphatidylinositol 3-kinase, inhibited flow-induced eNOS phosphorylation by 97% and NO production by 68%. Finally, PKB phosphorylated eNOS in vitro at the same site phosphorylated in the cell and increased eNOS enzymatic activity by 15-20-fold.
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PMID:Identification of flow-dependent endothelial nitric-oxide synthase phosphorylation sites by mass spectrometry and regulation of phosphorylation and nitric oxide production by the phosphatidylinositol 3-kinase inhibitor LY294002. 1051 97

Thrombopoietin (TPO), the critical regulator of platelet production, acts by binding to its cell surface receptor, c-Mpl. Numerous studies have shown that TPO binding leads to JAK2 kinase activation and Tyr phosphorylation of c-Mpl and several intracellular signaling intermediates, events vital for the biological activity of the hormone. In contrast, virtually nothing is known of the role of Ser or Thr phosphorylation of c-Mpl. By using phosphoamino acid analysis we found that Ser residues of c-Mpl were constitutively phosphorylated in receptor-bearing cells, levels that were increased following exposure of cells to TPO. To identify which residues were modified, and to determine the functional consequences of their phosphorylation, we generated a series of Ser to Ala mutations of a truncated c-Mpl receptor (T69) capable of supporting TPO-induced cell growth. Of the eight Ser within T69 we found that at least four are phosphorylated in TPO-stimulated cells. The mutation of each of these residues alone had minimal effects on TPO-induced proliferation, but substitution of all of the phosphoserine residues with Ala reduced the capacity of the receptor to support cell growth by over 50%. Additionally, the Ser at cytoplasmic position 18 is not detectably phosphorylated. However, the mutation of Ser-18 to Ala nearly abrogates TPO-induced proliferation and co-precipitation of JAK2 with Mpl. This study provides the first systematic analysis of the role of Ser residues in c-Mpl signaling.
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PMID:A structure-function analysis of serine/threonine phosphorylation of the thrombopoietin receptor, c-Mpl. 1091 61

Integrins are cell surface receptors for extracellular matrix, which play important roles in a variety of biological processes. 14-3-3 proteins are a highly conserved family of cytoplasmic proteins that associate with several intracellular signaling molecules in regulation of various cellular functions. Here, we report identification of an interaction between the integrin beta1 cytoplasmic domain and 14-3-3beta by using the yeast two-hybrid screen. Like several other proteins, the integrin beta1 cytoplasmic domain associated with 14-3-3beta by a non-phosphoserine mechanism. The 14-3-3beta/integrin beta1 interaction was confirmed by in vitro binding assays as well as co-precipitation in vivo. Furthermore, we found that 14-3-3beta co-localized with integrin beta1 during the early stage of cell spreading on fibronectin, suggesting a potential role of the 14-3-3beta/integrin beta1 interaction in the regulation of cell adhesion. Using tetracycline-regulated expression system, we showed that overexpression of 14-3-3beta stimulated cell spreading and migration on fibronectin but not on poly-L-lysine. However, the induced expression of 14-3-3beta did not affect tyrosine phosphorylation of FAK or its substrates, p130(cas) and paxillin, suggesting that 14-3-3beta regulated integrin-mediated cell spreading and migration by FAK-independent mechanisms. Taken together, these results identify an interaction between integrin and 14-3-3 proteins and suggest a potentially novel cellular function for 14-3-3 proteins in the regulation of integrin-mediated cell adhesion and signaling events.
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PMID:Identification of a novel interaction between integrin beta1 and 14-3-3beta. 1131 64

Megakaryocytic genes such as alphaIIbbeta3 can be expressed by malignant cells as part of the disturbances in their gene regulation. However, the function of the gene product greatly depends on the interaction of the ectopic protein with the new environment. The outside-in signaling of the ectopically expressed alphaIIbbeta3 integrin was studied in B16a murine melanoma cells using a monoclonal antibody, specifically directed to the activated conformation of alphaIIbbeta3, PAC-1 and the physiological ligand, fibrinogen. Ligation of alphaIIbbeta3 induced down-regulation of FAK but serine phosphorylation of three protein bands, 20/21, 85 and 140 kDa within 1-15 min. Flow cytometry indicated that the ligation of the receptor in B16a cells induces approximately 50% increase in phosphoserine positive cells within 5-15 min. 12-lipoxygenase is placed downstream in the signaling pathway, since ligation of alphaIIbbeta3 induces 12-HETE production within 5 min and pretreatment of tumor cells with select lipoxygenase inhibitior, Baicalein, prevents the increase in serine phosphorylation. Confocal microscopy of adherent tumor cells demonstrated rearrangement of actin filaments upon alphaIIbbeta3 ligation paralleled by downregulation of p125FAK and phoshotyrosine+ adhesion plaques and translocation of PKCalpha to stress fibers and cortical actin. PKC appears to be the major effector serine kinase of the alphaIIbbeta3-coupled signaling pathway, since pretreatment of tumor cells with a select PKC inhibitor, Calphostin C, prevents the ligation-induced serine phosphorylation. Previous studies have indicated a role for the 12-lipoxygenase-PKC signaling pathway in platelet aggregation as well as tumor invasion, therefore the involvement of this cascade in the signaling of the ectopic alphaIIbbeta3 integrin may partially explain its role in tumor progression.
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PMID:Ectopic alphaIIbbeta3 integrin signaling involves 12-lipoxygenase- and PKC-mediated serine phosphorylation events in melanoma cells. 1143 81

SRK is a plant receptor kinase involved in the self-incompatibility system of Brassica species. During a cDNA screening for the phosphoproteins from a stigma expression library, a clone encoding the nucleoside diphosphate kinase III (Bc-NDPK III) was obtained. After in vitro phosphorylation assays with recombinant proteins, Bc-NDPK III contained mostly phosphoserine. By contrast, the kinase domain of SRK contained phosphoserine and phosphothreonine, both of which were significantly increased by the addition of Bc-NDPK III in the presence of an SRK inhibitor KN-62. The result suggested the possible involvement of Bc-NDPK III in the signal transduction pathway through SRK.
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PMID:Isolation of a cDNA for a nucleoside diphosphate kinase capable of phosphorylating the kinase domain of the self-incompatibility factor SRK of Brassica campestris. 1188 97

The growth factor-activated AGC protein kinases RSK, S6K, PKB, MSK and SGK are activated by serine/threonine phosphorylation in the activation loop and in the hydrophobic motif, C-terminal to the kinase domain. In some of these kinases, phosphorylation of the hydrophobic motif creates a specific docking site that recruits and activates PDK1, which then phosphorylates the activation loop. Here, we discover a pocket in the kinase domain of PDK1 that recognizes the phosphoserine/phosphothreonine in the hydrophobic motif by identifying two oppositely positioned arginine and lysine residues that bind the phosphate. Moreover, we demonstrate that RSK2, S6K1, PKBalpha, MSK1 and SGK1 contain a similar phosphate-binding pocket, which they use for intramolecular interaction with their own phosphorylated hydrophobic motif. Molecular modelling and experimental data provide evidence for a common activation mechanism in which the phosphorylated hydrophobic motif and activation loop act on the alphaC-helix of the kinase structure to induce synergistic stimulation of catalytic activity. Sequence conservation suggests that this mechanism is a key feature in activation of >40 human AGC kinases.
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PMID:A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation. 1237 40


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