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)

In the present study we have investigated the effect of increased serine/threonine phosphorylation of insulin receptor substrates-1 and -2 (IRS-1 and IRS-2) by okadaic acid pretreatment on brown adipocyte insulin signalling leading to glucose transport, an important metabolic effect of insulin in brown adipose tissue. Okadaic acid pretreatment before insulin stimulation decreased IRS-1 and IRS-2 tyrosine phosphorylation in parallel to a decrease in their sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility. IRS-1/IRS-2-associated p85alpha and phosphatidylinositol (PI) 3-kinase enzymatic activity were partly reduced in brown adipocytes pretreated with okadaic acid upon stimulation with insulin. Furthermore, insulin-induced glucose uptake was totally abolished by the inhibitor in parallel with a total inhibition of insulin-induced protein kinase C (PKC) zeta activity. However, activation of Akt/PKB or p70 S6 kinase (p70(s6k)) by insulin remained unaltered. Our results suggest that downstream of PI 3-kinase, insulin signalling diverges into at least two independent pathways through Akt/PKB and PKC zeta, the PKC zeta pathway contributing to glucose transport induced by insulin in fetal brown adipocytes.
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PMID:Okadaic acid inhibits insulin-induced glucose transport in fetal brown adipocytes in an Akt-independent and protein kinase C zeta-dependent manner. 1078 24

Two protein-tyrosine kinases, Bruton's tyrosine kinase (Btk) and Syk, and members of the protein kinase C (PKC) subfamily of serine/threonine kinases play crucial roles in signal transduction through antigen receptors in B lymphocytes and high-affinity IgE receptors (FcepsilonRI) in mast cells. The present study provides genetic, biochemical, and pharmacological evidence that, on FcepsilonRI stimulation, Syk regulates Btk, and Btk selectively regulates the membrane translocation and enzymatic activity of PKCbetaI among the conventional PKC isoforms (alpha, betaI, and betaII) expressed in mast cells. Syk/Btk-mediated PKCbetaI regulation is involved in transcriptional activation of the IL-2 and tumor necrosis factor alpha genes through the JNK pathway induced by FcepsilonRI stimulation. Accordingly, FcepsilonRI-induced production of these cytokines is inhibited by specific inhibitors of Btk and Syk, as well as broad-specificity inhibitors of PKC and a selective inhibitor of PKCbeta. Specific regulation of PKCbetaI by Btk is consistent with the selective association of Btk with PKCbetaI. Components of this signaling pathway may represent an attractive set of potential targets of pharmaceutical interference for the treatment of allergic and other immunologic diseases.
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PMID:Regulation of protein kinase CbetaI by two protein-tyrosine kinases, Btk and Syk. 1085 54

The mechanism of outside-in signaling by integrins parallels that for growth factor receptors. In both pathways, phosphorylation of a cytoplasmic segment on tyrosine generates a docking site for proteins containing Src homology 2 (SH2) and phosphotyrosine binding domains. We recently observed that phosphorylation of a threonine (Thr-753), six amino acids proximal to tyrosine 759 in beta(3) of the platelet specific integrin alpha(IIb)beta(3), inhibits outside-in signaling through this receptor. We hypothesized that the presence of phosphothreonine 753 either renders beta(3) a poor substrate for tyrosine kinases or inhibits the docking capabilities of the tyrosyl-phosphorylated form of beta(3.) The first alternative was tested by comparing the phosphorylation of beta(3) model peptides by the tyrosine kinase pp60(c-src) and we found that the presence of a phosphate group on a residue corresponding to Thr-753 did not detectably alter the kinetics of tyrosine phosphorylation. However, the presence of phosphate on this threonine inhibited the binding of Shc to tyrosyl-phosphorylated beta(3) peptide. The inhibitory effect of the phosphate group could be mimicked by substituting an aspartic acid for Thr-753, suggesting that a negative charge at this position modulates the binding of Shc and possibly other phosphotyrosine binding domain- and SH2-containing proteins. A survey of several protein kinases revealed that Thr-753 was avidly phosphorylated by PDK1 and Akt/PKB in vitro. These observations suggest that activation of PDK1 and/or Akt/PKB in platelets may modulate the binding activity and/or specificity of beta(3) for signaling molecules.
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PMID:Threonine phosphorylation of the beta 3 integrin cytoplasmic tail, at a site recognized by PDK1 and Akt/PKB in vitro, regulates Shc binding. 1089 34

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is important for the regulation of a number of cellular responses. Serine/threonine kinase Akt (protein kinase B; PKB) is downstream of PI3K and activated by growth factors. This study found that erythropoietin (EPO) induced tyrosine phosphorylation of Akt in a time- and dose-dependent manner in EPO-dependent human leukemia cell line UT-7/EPO. In vitro kinase assay using histone H2B and glucose synthase kinase as substrates demonstrated that Akt was actually activated by EPO. EPO-induced phosphorylation of Akt was completely blocked by a PI3K-specific inhibitor, LY294002, at 10 micromol/L, indicating that activation of Akt by EPO is dependent on PI3K activity. In addition, overexpression of the constitutively active form of Akt on UT-7/EPO cells partially blocked apoptosis induced by withdrawal of EPO from the culture medium. This finding suggested that the PI3K-Akt activation pathway plays some role in the antiapoptotic effect of EPO. EPO induced phosphorylation of a member of the trancription factor Forkhead family, FKHRL1, at threonine 32 and serine 253 in a dose- and time-dependent manner in UT-7/EPO cells. Moreover, results showed that Akt kinase activated by EPO directly phosphorylated FKHRL1 protein and that FKHRL1 phosphorylation was completely dependent on PI3K activity as is the case for Akt. In conjunction with the evidence that FKHRL1 is expressed in normal human erythroid progenitor cells and erythroblasts, the results suggest that FKHRL1 plays an important role in erythropoiesis as one of the downstream target molecules of PI3K-Akt.
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PMID:A member of Forkhead family transcription factor, FKHRL1, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt activation pathway in erythropoietin signal transduction. 1091 Sep 8

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

Suppressor of cytokine signaling (SOCS) family proteins were originally identified as cytokine-induced negative regulators of cytokine signaling. We show that SOCS-1 and SOCS-3 inhibit interleukin (IL)-4-dependent signal transducer and activator of transcription 6 (Stat6) activation of and subsequent gene induction. By contrast, SOCS-2 and cytokine-inducible Src homology domain 2 (SH2)-containing protein up-regulate these processes. IL-4 initiates transmembrane signaling through two types of receptor complexes comprising the IL-4Ralpha subunit and the associated Janus kinase 1 (Jak1) as common essential components. We demonstrate that both SOCS-1- and SOCS-3-mediated down-regulation of IL-4 signaling is due to an inhibition of the receptor associated Jak1 activity. The SOCS proteins contain an amino-terminal region of variable length and primary structure, a central SH2 domain, and a carboxyl-terminal conserved motif termed SOCS-box. We show that the SH2 domains of SOCS-2, SOCS-3, and cytokine-inducible SH2-containing protein are functionally redundant in regulating the IL-4-dependent Jak-Stat signaling. The Pre-SH2 domains of SOCS-2 and SOCS-3 confer the specificity of their regulatory function. Importantly, the Pre-SH2 domain of SOCS-3 alone can inhibit IL-4 signaling. The SH2-proximal 25 amino acids of SOCS-3 are sufficient for this inhibition, and the Thr residue at position 24 and the Phe residue at position 25 are individually indispensable for its inhibitory function. Thus, the Thr-Phe motif in the Pre-SH2 domain plays a critical role in SOCS-3-mediated inhibition of the IL-4-dependent Jak-Stat signaling, likely by regulating the mode of SOCS-Jak interaction.
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PMID:Identification of critical residues required for suppressor of cytokine signaling-specific regulation of interleukin-4 signaling. 1095 Sep 67

Adhesion stabilization is a prerequisite for the long-term adhesion of circulating metastatic tumor cells, and tumor cells with different metastatic potential demonstrate distinct patterns of cell adhesion properties. An important event during formation of organ metastases is integrin-mediated extracellular matrix (ECM) binding that can initiate signal transduction events. Recently we reported that Ser/Thr kinases are involved in regulation of tumor cell adhesion. In the present study the influence of dephosphorylation by Ser/Thr protein phosphatases (PPases) on tumor cell adhesion was investigated. Pretreatment of poorly and highly metastatic human HT-29 colon carcinoma cells with the broad-range inhibitors sodium fluoride (NaF) and sodium pyrophosphate (PyroP) resulted in strong reduction in adhesion of HT-29 cells to various ECM components. Surprisingly, when specific Ser/Thr PPase inhibitors like tautomycin were used we found only a partial reduction in adhesion of highly metastatic HT-29LMM cells to collagen I but not to collagen IV. Other inhibitors did not inhibit adhesion, and poorly metastatic HT-29P were not affected by any specific Ser/Thr PPase inhibitors. Therefore, the effects of NaF on adhesion-mediated Tyr phosphorylation were investigated further. Pretreatment with this inhibitor led to a reduction in phosphorylation of focal adhesion kinase (FAK). In contrast, in cells grown adherent to tissue culture dishes, low concentrations of NaF increased FAK phosphorylation whereas high concentrations inhibited the amount of phosphorylated FAK. Although NaF inhibited adhesions it did not cause changes in cell morphology or detachment of cells from ECM. We hypothesize that dual-specific PPases may be involved in the regulation and establishment of new adhesive interactions in HT-29 cells, but they are not required for maintenance of stable adhesions to ECM.
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PMID:Time-dependent dephosphorylation through serine/threonine phosphatases is required for stable adhesion of highly and poorly metastatic HT-29 colon carcinoma cell lines to collagen. 1095 84

Serine/threonine kinase Akt/PKB is a downstream effector molecule of phosphoinositide 3-kinase and is thought to mediate many biological actions toward anti-apoptotic responses. We found that Akt formed a complex with a 90-kDa heat-shock protein (Hsp90) in vivo. By constructing deletion mutants, we identified that amino acid residues 229-309 of Akt were involved in the binding to Hsp90 and amino acid residues 327-340 of Hsp90beta were involved in the binding to Akt. Inhibition of Akt-Hsp90 binding led to the dephosphorylation and inactivation of Akt, which increased sensitivity of the cells to apoptosis-inducing stimulus. The dephosphorylation of Akt was caused by an increase in protein phosphatase 2A (PP2A)-mediated dephosphorylation and not by a decrease in 3-phosphoinositide-dependent protein kinase-1-mediated phosphorylation. These results indicate that Hsp90 plays an important role in maintaining Akt kinase activity by preventing PP2A-mediated dephosphorylation.
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PMID:Modulation of Akt kinase activity by binding to Hsp90. 1099 57

Phosphorylation of Thr(308) in the activation loop and Ser(473) at the carboxyl terminus is essential for protein kinase B (PKB/Akt) activation. However, the biochemical mechanism of the phosphorylation remains to be characterized. Here we show that expression of a constitutively active mutant of mouse 3-phosphoinositide-dependent protein kinase-1 (PDK1(A280V)) in Chinese hamster ovary cells overexpressing the insulin receptor was sufficient to induce PKB phosphorylation at Thr(308) to approximately the same extent as insulin stimulation. Phosphorylation of PKB by PDK1(A280V) was not affected by treatment of cells with inhibitors of phosphatidylinositol 3-kinase or by deletion of the pleckstrin homology (PH) domain of PKB. C(2)-ceramide, a cell-permeable, indirect inhibitor of PKB phosphorylation, did not inhibit PDK1(A280V)-catalyzed PKB phosphorylation in cells and had no effect on PDK1 activity in vitro. On the other hand, co-expression of full-length protein kinase C-related kinase-1 (PRK1/PKN) or 2 (PRK2) inhibited PDK1(A280V)-mediated PKB phosphorylation. Replacing alanine at position 280 with valine or deletion of the PH domain enhanced PDK1 autophosphorylation in vitro. However, deletion of the PH domain of PDK1(A280V) significantly reduced PDK1(A280V)-mediated phosphorylation of PKB in cells. In resting cells, PDK1(A280V) localized in the cytosol and at the plasma membrane. However, PDK1(A280V) lacking the PH domain localized predominantly in the cytosol. Taken together, our findings suggest that the wild-type PDK1 may not be constitutively active in cells. In addition, activation of PDK1 is sufficient to phosphorylate PKB at Thr(308) in the cytosol. Furthermore, the PH domain of PDK1 may play both positive and negative roles in regulating the in vivo function of the enzyme. Finally, unlike the carboxyl-terminal fragment of PRK2, which has been shown to bind PDK1 and allow the enzyme to phosphorylate PKB at both Thr(308) and Ser(473), full-length PRK2 and its related kinase PRK1/PKN may both play negative roles in PKB-mediated downstream biological events.
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PMID:Mechanism of phosphorylation of protein kinase B/Akt by a constitutively active 3-phosphoinositide-dependent protein kinase-1. 1100 71

Integrin-mediated cell adhesion is known to regulate gene expression through the activation of transcription factors. We have recently revealed that these activations are mediated through integrin-linked kinase (ILK). ILK is an ankyrin repeat-containing serine-threonine protein kinase that can interact directly with the cytoplasmic domain of the beta1 and beta3 integrin subunits and whose kinase activity is modulated by cell-extracellular matrix interactions. We have shown that ILK overexpression results in the translocation of beta-catenin to the nucleus, which then forms a complex formation with the lymphoid enhancer binding factor 1 (LEF-1) transcription factor, subsequently activating the transcriptional activity of promoters containing LEF-1 response elements. ILK phosphorylates the glycogen synthase kinase-3 (GSK-3), which inhibits GSK-3 activity. We have demonstrated that ILK stimulates activator protein-1 transcriptional activity through GSK-3 and the subsequent regulation of the c-Jun-DNA interaction. ILK also phosphorylates protein kinase B (PKB/Akt) and stimulates its activity. We have shown that ILK is an upstream effector of the phosphatidylinositol 3-kinase-dependent regulation of PKB/Akt. ILK has been shown to phosphorylate PKB/Akt on Ser-473 in vitro and in vivo. Our results clearly indicate that ILK is a key element in the regulation of integrin signaling as well as growth factor and Wnt signaling pathways. PTEN (phosphatase and tensin homolog detected on chromosome 10) is a tumor suppressor gene located on chromosome 10q23 that encodes a protein and phospholipid phosphatase. It is now estimated that inactivation mutants of PTEN exist in 60% of all forms of solid tumors. Loss of expression or mutational inactivation of PTEN leads to the constitutive activation of PKB/Akt via enhanced phosphorylation of Thr-308 and Ser-473. We have demonstrated that the activity of ILK is constitutively elevated in PTEN mutant cells. A small molecule ILK inhibitor suppresses the phosphorylation of PKB at the Ser-473 but not the Thr-308 site in the PTEN mutant cells. These results indicate that inhibition of ILK may be of significant value in solid tumor therapy.
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PMID:Integrin-linked kinase (ILK): a "hot" therapeutic target. 1100 49


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