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)

PKB/Akt, S6K1 and SGK are related protein kinases activated in a PI 3-kinase-dependent manner in response to insulin/growth factors signalling. Activation entails phosphorylation of these kinases at two residues, the T-loop and the hydrophobic motif. PDK1 activates S6K, SGK and PKB isoforms by phosphorylating these kinases at their T-loop. We demonstrate that a pocket in the kinase domain of PDK1, termed the 'PIF-binding pocket', plays a key role in mediating the interaction and phosphorylation of S6K1 and SGK1 at their T-loop motif by PDK1. Our data indicate that prior phosphorylation of S6K1 and SGK1 at their hydrophobic motif promotes their interaction with the PIF-binding pocket of PDK1 and their T-loop phosphorylation. Thus, the hydrophobic motif phosphorylation of S6K and SGK converts them into substrates that can be activated by PDK1. In contrast, the PIF-binding pocket of PDK1 is not required for the phosphorylation of PKBalpha by PDK1. The PIF-binding pocket represents a substrate recognition site on a protein kinase that is only required for the phosphorylation of a subset of its physiological substrates.
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PMID:The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB. 1150 Mar 65

In the rat corpus luteum of pregnancy, PRL stimulation of ER expression is a prerequisite for E2 to have any luteotropic effect. Previous work from our laboratory has established that PRL stimulates ERalpha expression at the level of transcription and that the transcription factor Stat5 (signal transducer and activator of transcription 5) mediates this stimulation. Since it is well established that PRL activates Stat5 through the tyrosine kinase, Janus kinase 2 (Jak2), the role of Jak2 in PRL regulation of ERalpha expression was investigated. In primary luteinized granulosa cells, the general tyrosine kinase inhibitors, genistein and AG18, and the Jak2 inhibitor, AG490, prevented PRL stimulation of ERalpha mRNA levels, suggesting that PRL signaling to the ERalpha gene requires Jak2 activity. However, using an antibody that recognizes the tyrosine-phosphorylated forms of both Stat5a and Stat5b (Y694/Y699), it was found that AG490 could inhibit PRL-induced Stat5a phosphorylation only and had little or no effect on Stat5b phosphorylation. These effects of AG490 were confirmed in COS cells overexpressing Stat5b. Also in COS cells, a kinase-negative Jak2 prevented PRL stimulation of ERalpha promoter activity and Stat5b phosphorylation while a constitutively active Jak2 could stimulate both in the absence of PRL. Furthermore, kinase-negative-Jak2, but not AG490, could inhibit Stat5b nuclear translocation and DNA binding. Therefore, it seems that in the presence of AG490, Stat5b remains phosphorylated, is located in the nucleus and capable of binding DNA, but is apparently transcriptionally inactive. These findings suggest that PRL may activate a second tyrosine kinase, other than Jak2, that is capable of phosphorylating Stat5b without inducing transcriptional activity. To investigate whether another signaling pathway is involved, the src kinase inhibitor PP2 and the phosphoinositol-3 kinase inhibitor (PI3K), LY294002, were used. Neither inhibitor alone had any major effect on PRL regulation of ERalpha promoter activity or on PRL-induced Stat5b phosphorylation. However, the combination of AG490 and LY294002 largely prevented PRL-induced Stat5b phosphorylation. These findings indicate that PRL stimulation of ERalpha expression requires Jak2 and also that PRL can induce Stat5b phosphorylation through two tyrosine kinases, Jak2 and one downstream of PI3K. Furthermore, these results suggest that the role of Jak2 in activating Stat5b may be through a mechanism other than simply inducing Stat5b phosphorylation.
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PMID:PRL-induced ERalpha gene expression is mediated by Janus kinase 2 (Jak2) while signal transducer and activator of transcription 5b (Stat5b) phosphorylation involves Jak2 and a second tyrosine kinase. 1168 25

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in activating the AGC subfamily of protein kinases. In particular, PDK1 plays an important role in the regulation of Akt/PKB survival pathway by phosphorylating Akt on Thr308. Here we show that UCN-01 (7-hydroxystaurosporine), a drug now in clinical trials and with a unique fingerprint pattern, induced dephosphorylation and inactivation of Akt, resulting in the turn-off of the survival signals and the induction of apoptosis. Further analysis revealed that UCN-01-mediated Akt inactivation was caused by inhibiting upstream Akt kinase PDK1 (IC50=33 nM) both in vitro and from cells, but not by suppressing Akt itself or phosphatidylinositide-3-OH kinase. UCN-01-induced PDK1 inhibition was also observed in in vivo murine and human tumor xenografts. Overexpression of active form of Akt diminished the cytotoxic effects of UCN-01, suggesting that UCN-01 may in part exert its cytotoxicity by inhibiting PDK1-Akt survival pathway. Because UCN-01 has already proved to have potent anti-tumor activity in vivo, PDK1-Akt survival pathway is a new, attractive target for cancer chemotherapy.
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PMID:Interference with PDK1-Akt survival signaling pathway by UCN-01 (7-hydroxystaurosporine). 1189 4

PDK1 functions as a master kinase, phosphorylating and activating PKB/Akt, S6K and RSK. To learn more about the roles of PDK1, we generated mice that either lack PDK1 or possess PDK1 hypomorphic alleles, expressing only approximately 10% of the normal level of PDK1. PDK1(-/-) embryos die at embryonic day 9.5, displaying multiple abnormalities including lack of somites, forebrain and neural crest derived tissues; however, development of hind- and midbrain proceed relatively normally. In contrast, hypomorphic PDK1 mice are viable and fertile, and insulin injection induces the normal activation of PKB, S6K and RSK. Nevertheless, these mice are 40-50% smaller than control animals. The organ volumes from the PDK1 hypomorphic mice are reduced proportionately. We also establish that the volume of a number of PDK1-deficient cells is reduced by 35-60%, and show that PDK1 deficiency does not affect cell number, nuclear size or proliferation. We provide genetic evidence that PDK1 is essential for mouse embryonic development, and regulates cell size independently of cell number or proliferation, as well as insulin's ability to activate PKB, S6K and RSK.
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PMID:Essential role of PDK1 in regulating cell size and development in mice. 1211 May 85

The protein kinase Akt/PKB is stimulated by the phosphorylation of two regulatory residues, Thr 309 of the activation segment and Ser 474 of the hydrophobic motif (HM), that are structurally and functionally conserved within the AGC kinase family. To understand the mechanism of PKB regulation, we determined the crystal structures of activated kinase domains of PKB in complex with a GSK3beta-peptide substrate and an ATP analog. The activated state of the kinase was generated by phosphorylating Thr 309 using PDK1 and mimicking Ser 474 phosphorylation either with the S474D substitution or by replacing the HM of PKB with that of PIFtide, a potent mimic of a phosphorylated HM. Comparison with the inactive PKB structure indicates that the role of Ser 474 phosphorylation is to promote the engagement of the HM with the N-lobe of the kinase domain, promoting a disorder-to-order transition of the alphaC helix. The alphaC helix, by interacting with pThr 309, restructures and orders the activation segment, generating an active kinase conformation. Analysis of the interactions between PKB and the GSK3beta-peptide explains how PKB selects for protein substrates distinct from those of PKA.
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PMID:Crystal structure of an activated Akt/protein kinase B ternary complex with GSK3-peptide and AMP-PNP. 1243 48

A high-affinity IL-2 receptor requires two Janus protein tyrosine kinases (JAKs) for IL-2 signal transduction: JAK1 and JAK3. Since transphosphorylation of the two kinases is presumed to occur after receptor engagement we examined the phosphorylation by recombinant JAK3 of a peptide substrate corresponding to the JAK1 activation loop (KAIETDKEYYTVKD), which has two adjacent tyrosines. Mass spectral analysis of the enzymatically phosphorylated peptide showed that the second tyrosine was phosphorylated at a 30-fold greater rate than the first tyrosine. Moreover, no doubly phosphorylated peptide was detected by this analysis. Kinetic analysis of the reactions of singly phosphorylated JAK1 activation loop peptides showed that phosphorylating the first or second tyrosine decreased the k(cat)/K(m) for the phosphorylation of the other 115- and 26-fold, respectively. Singly changing each side chain of the KEYYTV portion of the peptide to a methyl group (alanine) yielded substrates comparable to the wild-type sequences in all cases except that of the first or second tyrosine, which showed a 153- or 70-fold drop in k(cat)/K(m), respectively. Using libraries of immobilized peptides with all 20 naturally occurring amino acids substituted for Y9 or T11 showed that the JAK3 tolerated substitution at T11 but prefers large hydrophobic amino acids at Y9. These results show that JAK3 does not act processively on the JAK1 activation loop in vitro and illustrate the role of Y9 in the recognition of the preferred site of phosphorylation which is Y10.
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PMID:Mechanism of Janus kinase 3-catalyzed phosphorylation of a Janus kinase 1 activation loop peptide. 1255 72

CSK family contains two protein tyrosine kinases: Csk (C-terminal Src kinase) and Chk (Csk homologous kinase). They are responsible for phosphorylating Src family protein tyrosine kinases on a C-terminal Tyr (Tyr527) and negatively regulating their activities. However, Chk and Csk have different expression patterns, mechanisms of regulation, and different biological functions, and appear to play different roles in the development of breast cancer. To obtain pure human Chk for biochemical characterization, its coding region was amplified by polymerase chain reaction and expressed as a fusion protein with glutathione S-transferase in Escherichia coli. The enzyme was highly expressed but unusually prone to proteolytic degradation during purification. Expression of the enzyme as a dual fusion protein with glutathione S-transferase on N-terminus and streptag, a 10 amino acid peptide, on C-terminus allowed purification of the full-length fusion protein. The purified enzyme was able to phosphorylate and inactivate Src. Chk (no inhibition up to 18.5 microM) and Csk (IC(50)= 1 microM) were differentially inhibited by PP2, probably due to the size difference of one residue (Thr265 in Csk versus Met304 in Chk) in the ATP-binding domain. The expression, purification, and initial characterizations of Chk provided an important step toward full characterization of Chk and Csk, two important enzymes in cellular regulation.
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PMID:Expression, purification, and biochemical characterization of Chk, a soluble protein tyrosine kinase. 1276 3

Cbp, a C-terminal Src kinase (Csk)-binding protein, is a transmembrane phosphoprotein that has been implicated in the regulation of the Src family kinase (SFK) through recruiting Csk, a negative regulator of SFK, to a membrane microdomain of lipid rafts. To examine the contribution of Cbp to cell adhesion signaling mediated by SFK, we investigated the kinase responsible for phosphorylating Cbp and the mode of phosphorylation during the cell adhesion process. The results obtained by using mutant mice or cells that lack Csk and/or a member of SFK, Fyn, reveal that Cbp is phosphorylated predominantly by raft-localized Fyn in vivo. Upon cell adhesion onto fibronectin, Cbp becomes transiently phosphorylated (consistent with SFK activation) and recruits Csk to lipid rafts. These events are completed before the full activation of focal adhesion kinase, indicating that the transient activation and down-regulation of SFK in lipid rafts are earlier events in cell adhesion signaling. In Csk-deficient cells, continuous hyperactivation of SFK leads to continuous hyperphosphorylation of Cbp, accompanied by impaired cell spreading and migration. Silencing of Cbp by RNA interference also induced impaired cell spreading. These findings suggest that Cbp could serve as a sensor of SFK activity in early stages of cell adhesion signaling, and that Csk-mediated down-regulation of SFK is essential to allow dynamic cellular events involved in the regulation of cell spreading and migration.
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PMID:Transmembrane phosphoprotein Cbp senses cell adhesion signaling mediated by Src family kinase in lipid rafts. 1464 15

Insulin stimulates muscle glucose disposal via both glycolysis and glycogen synthesis. Insulin activates glycogen synthase (GS) in skeletal muscle by phosphorylating PKB (or Akt), which in turn phosphorylates and inactivates glycogen synthase kinase 3 (GSK-3), with subsequent activation of GS. A rapamycin-sensitive pathway, most likely acting via ribosomal 70-kDa protein S6 kinase (p70(S6K)), has also been implicated in the regulation of GSK-3 and GS by insulin. Amino acids potently stimulate p70(S6K), and recent studies on cultured muscle cells suggest that amino acids also inactivate GSK-3 and/or activate GS via activating p70(S6K). To assess the physiological relevance of these findings to normal human physiology, we compared the effects of amino acids and insulin on whole body glucose disposal, p70(S6K), and GSK-3 phosphorylation, and on the activity of GS in vivo in skeletal muscle of 24 healthy human volunteers. After an overnight fast, subjects received intravenously either a mixed amino acid solution (1.26 micromol.kg(-1).min(-1) x 6 h, n = 9), a physiological dose of insulin (1 mU.kg(-1).min(-1) euglycemic hyperinsulinemic clamp x 2 h, n = 6), or a pharmacological dose of insulin (20 mU.kg(-1).min(-1) euglycemic hyperinsulinemic clamp x 2 h, n = 9). Whole body glucose disposal rates were assessed by calculating the steady-state glucose infusion rates, and vastus lateralis muscle was biopsied before and at the end of the infusion. Both amino acid infusion and physiological hyperinsulinemia enhanced p70(S6K) phosphorylation without affecting GSK-3 phosphorylation, but only physiological hyperinsulinemia also increased whole body glucose disposal and GS activity. In contrast, a pharmacological dose of insulin significantly increased whole body glucose disposal, p70(S6K), GSK-3 phosphorylation, and GS activity. We conclude that amino acids at physiological concentrations mediate p70(S6K) but, unlike insulin, do not regulate GSK-3 and GS phosphorylation/activity in human skeletal muscle.
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PMID:Unlike insulin, amino acids stimulate p70S6K but not GSK-3 or glycogen synthase in human skeletal muscle. 1465 17

Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. Unlike other Cdks that promote cell cycle, Cdk5 is activated in postmitotic neurons and critically regulates neuronal migration by phosphorylating its substrates during brain development. Recently, we found that Cdk5 phosphorylates focal adhesion kinase (FAK) at Serine 732 in vitro and is responsible for this phosphorylation in the developing brain. Our experiments using a phospho-specific antibody and an S732-unphosphorylatable mutant FAK suggest that S732 phosphorylation may regulate a centrosome-associated microtubule structure to promote nuclear translocation, a critical step in neuronal migration. S732 phosphorylation does not directly impact on the kinase activity of FAK, but appears to prevent the accumulation of FAK at the centrosome. Our study reveals a similarity between Cdk5 and Cdk1 in the regulation of neuronal migration and cell division, respectively. In addition, our study implicates FAK in a signaling pathway that directly regulates microtubules.
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PMID:Cdk5 phosphorylation of FAK regulates centrosome-associated miocrotubules and neuronal migration. 1471 65


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