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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Okadaic acid (OA) is a potent inhibitor of PP1 and PP2A serine/threonine phosphatases and an inhibitor of phosphatidylinositol 3'-kinase (PI 3-kinase) recruitment/ activation. Here we report that PI 3-kinase associates with a serine kinase activated by OA. Whole cell phosphorylation studies showed that PI 3-kinase associates with a wortmannin insensitive 76 kDa serine phosphoprotein (pp76) distinct from the p85 subunit of PI 3-kinase. Serine kinase assays demonstrated that pp76 phosphorylation was dependent upon a wortmannin insensitive serine kinase contained within PI 3-kinase/pp76 complexes and that this kinase had different cation requirements than PI 3-kinase serine kinase. Treatment of whole cells with OA lead to a wortmannin-independent 7.6-fold increase in pp76 serine phosphorylation and to a 7-fold rise in pp76 kinase activity. Together, these findings indicate that pp76 is a PI 3-kinase associated phosphoprotein and suggest that pp76 may be a novel PI 3-kinase associated serine kinase that is activated by OA.
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PMID:Phosphatidylinositol 3'-kinase is associated with a serine kinase that is activated by okadaic acid. 946 47

Serine phosphorylation of insulin receptor substrate-1 (IRS-1) has been proposed as a counter-regulatory mechanism in insulin and cytokine signalling. Here we report that IRS-1 is phosphorylated by a wortmannin insensitive phosphatidylinositol 3'-kinase (PI 3-kinase)-associated serine kinase (PAS kinase) distinct from PI 3-kinase serine kinase. We found that PI 3-kinase immune complexes contain 5-fold more wortmannin-insensitive serine kinase activity than SH2-containing protein tyrosine phosphatase-2 (SHP2) and IRS-1 immune complexes. Affinity chromatography of cell lysates with a glutathione S-transferase fusion protein for the p85 subunit of PI 3-kinase showed that PAS kinase associated with the p85 subunit of PI 3-kinase. This interaction required unoccupied SH2 domain(s) but did not require the PI 3-kinase p110 subunit binding domain. In terms of function, PAS kinase phosphorylated IRS-1 and, after insulin stimulation, PAS kinase phosphorylated IRS-1 in PI 3-kinase-IRS-1 complexes. Phosphopeptide mapping showed that insulin-dependent in vivo sites of IRS-1 serine phosphorylation were comparable to those of PAS kinase phosphorylated IRS-1. More importantly, PAS kinase-dependent phosphorylation of IRS-1 reduced by 4-fold the ability of IRS-1 to act as an insulin receptor substrate. Taken together, these findings indicate that: (a) PAS kinase is distinct from the intrinsic serine kinase activity of PI 3-kinase, (b) PAS kinase associates with the p85 subunit of PI 3-kinase through SH2 domain interactions, and (c) PAS kinase is an IRS-1 serine kinase that can reduce the ability of IRS-1 to serve as an insulin receptor substrate.
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PMID:Phosphatidylinositol 3'-kinase associates with an insulin receptor substrate-1 serine kinase distinct from its intrinsic serine kinase. 976 40

Both p21ras and phosphatidylinositol 3-kinase (PI 3-k) are critical elements in signaling pathways mediating insulin/IGF-I induced cell cycle progression. For example, microinjection of antibodies, peptides, or recombinant proteins which block the interaction of the SH2 domains of the PI 3-k p85alpha subunit with tyrosine phosphorylated intracellular targets blocks insulin mediated DNA synthesis. We report here that this inhibitory phenotype is observed whether the injections are made into quiescent cells (the standard approach), or at any time point during G1 phase subsequent to stimulation. This observation is not true, however, for the major substrate of the insulin/IGF-I receptor (IRS-1) despite the well known interaction of p85 with IRS-1. Antibodies to IRS-1 are inhibitory only when injected during the first 15 min of G1 phase, as are antibodies to another major IRS-1 binding protein, the tyrosine phosphatase SHP2. We also have microinjected reagents which target proteins involved in the formation of rasGTP and which mediate some of the downstream effects of ras activation. Reagents which target the formation of rasGTP (Shc and dominant negative ras protein) inhibit DNA synthesis only at points early in G1, as do reagents which target components of the MAP kinase pathway. Injection of antibodies to p21ras itself, or a recombinant Raf-1 protein domain which binds to the effector region of ras in a GTP-dependent manner, results in the inhibition of cell cycle progression throughout G1 phase. The results point to a continuous requirement for both PI 3-k and ras activity until cellular commitment to DNA synthesis, although some of the molecules which are both upstream and downstream of these activities are only required transiently. Our results are also consistent with a Raf-1 independent ras activity late in G1, as well as IRS-1 independent effects of PI 3-kinase.
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PMID:Prolonged vs transient roles for early cell cycle signaling components. 978 5

Vanadate and peroxovanadate (pV), potent inhibitors of tyrosine phosphatases, mimic several of the metabolic actions of insulin. Here we compare the mechanisms for the anti-lipolytic action of insulin, vanadate and pV in rat adipocytes. Vanadate (5 mM) and pV (0.01 mM) inhibited lipolysis induced by 0.01-1 microM isoprenaline, vanadate being more and pV less efficient than insulin (1 nM). A loss of anti-lipolytic effect of pV was observed by increasing the concentration of isoprenaline and/or pV. pV induced tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 to a greater extent than insulin, whereas vanadate affected these components little if at all. In addition, only a higher concentration (0.1 mM) of pV induced the tyrosine phosphorylation of p85, the 85 kDa regulatory subunit of phosphoinositide 3-kinase (PI-3K). Vanadate activated PI-3K-independent (in the presence of 10 nM isoprenaline) and PI-3K-dependent (in the presence of 100 nM isoprenaline) anti-lipolytic pathways, both of which were found to be independent of phosphodiesterase type 3B (PDE3B). pV (0.01 mM), like insulin, activated PI-3K- and PDE3B-dependent pathways. However, the anti-lipolytic pathway of 0.1 mM pV did not seem to require insulin receptor substrate-1-associated PI-3K and was found to be partly independent of PDE3B. Vanadate and pV (only at 0.01 mM), like insulin, decreased the isoprenaline-induced activation of cAMP-dependent protein kinase. Overall, these results underline the complexity and the diversity in the mechanisms that regulate lipolysis.
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PMID:Mechanisms of inhibition of lipolysis by insulin, vanadate and peroxovanadate in rat adipocytes. 1019 Dec 58

The work of Reddy et al. (S. A. Reddy, J. A. Huang, and W. S. Liao, J. Biol. Chem. 272:29167-29173, 1997) reveals that phosphatidylinositol 3-kinase (PI3K) plays a role in transducing a signal from the occupied interleukin-1 (IL-1) receptor to nuclear factor kappaB (NF-kappaB), but the underlying mechanism remains to be determined. We have found that IL-1 stimulates interaction of the IL-1 receptor accessory protein with the p85 regulatory subunit of PI3K, leading to the activation of the p110 catalytic subunit. Specific PI3K inhibitors strongly inhibit both PI3K activation and NF-kappaB-dependent gene expression but have no effect on the IL-1-stimulated degradation of IkappaBalpha, the nuclear translocation of NF-kappaB, or the ability of NF-kappaB to bind to DNA. In contrast, PI3K inhibitors block the IL-1-stimulated phosphorylation of NF-kappaB itself, especially the p65/RelA subunit. Furthermore, by using a fusion protein containing the p65/RelA transactivation domain, we found that overexpression of the p110 catalytic subunit of PI3K induces p65/RelA-mediated transactivation and that the specific PI3K inhibitor LY294,002 represses this process. Additionally, the expression of a constitutively activated form of either p110 or the PI3K-activated protein kinase Akt also induces p65/RelA-mediated transactivation. Therefore, IL-1 stimulates the PI3K-dependent phosphorylation and transactivation of NF-kappaB, a process quite distinct from the liberation of NF-kappaB from its cytoplasmic inhibitor IkappaB.
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PMID:Activation of phosphatidylinositol 3-kinase in response to interleukin-1 leads to phosphorylation and activation of the NF-kappaB p65/RelA subunit. 1037 29

ERM (Ezrin-Radixin-Moesin) proteins function as plasma membrane-actin cytoskeleton linkers and participate in the formation of specialized domains of the plasma membrane. We have investigated ezrin function in tubulogenesis of a kidney-derived epithelial cell line, LLC-PK1. Here we show that cells overproducing a mutant form of ezrin in which Tyr-353 was changed to a phenylalanine (Y353F) undergo apoptosis when assayed for tubulogenesis. While investigating the mechanism responsible for this apoptosis, we found that ezrin interacts with p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase). Two distinct sites of ezrin are involved in this interaction, the amino-terminal domain containing the first 309 aa and the phosphorylated Tyr-353 residue, which binds to the carboxyl-terminal SH2 domain of p85. Cells producing Y353F ezrin are defective in activation of the protein kinase Akt, a downstream target of PI 3-kinase that protects cells against apoptosis. Furthermore, the apoptotic phenotype of these cells is rescued by production of a constitutively activated form of PI 3-kinase. Taken together, these results establish a novel function for ezrin in determining survival of epithelial cells by activating the PI 3-kinase/Akt pathway.
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PMID:Ezrin, a plasma membrane-microfilament linker, signals cell survival through the phosphatidylinositol 3-kinase/Akt pathway. 1037 9

We have employed C2C12 myotubes to investigate lipid inhibition of insulin-stimulated signal transduction and glucose metabolism. Cells were preincubated for 18 h in the absence or presence of free fatty acids (FFAs) and stimulated with insulin, and the effects on glycogen synthesis and signaling intermediates were determined. While the unsaturated FFAs oleate and linoleate inhibited both basal and insulin-stimulated glycogen synthesis, the saturated FFA palmitate reduced only insulin-stimulated glycogen synthesis, and was found to inhibit insulin-stimulated phosphorylation of glycogen synthase kinase-3 and protein kinase B (PKB). However, no effect of palmitate was observed on tyrosine phosphorylation, p85 association, or phosphatidylinositol 3-kinase activity in IRS-1 immunoprecipitates. In contrast, palmitate promoted phosphorylation of mitogen-activated protein MAP) kinases. Ceramide, a derivative of palmitate, has recently been associated with similar inhibition of PKB, and here, ceramide levels were found to be elevated 2-fold in palmitate-treated C2C12 cells. Incubation of C2C12 cells with ceramide closely reproduced the effects of palmitate, leading to inhibition of glycogen synthesis and PKB and to stimulation of MAP kinase. We conclude that palmitate-induced insulin resistance occurs by a mechanism distinct from that of unsaturated FFAs, and involves elevation of ceramide by de novo synthesis, leading to PKB inhibition without affecting IRS-1 function.
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PMID:Ceramide generation is sufficient to account for the inhibition of the insulin-stimulated PKB pathway in C2C12 skeletal muscle cells pretreated with palmitate. 1044 95

The effects of cyclic AMP (cAMP) on cell proliferation are cell type specific. Although the growth-inhibitory effects of cAMP have been well studied, much less is known regarding how cAMP stimulates proliferation. We report that cAMP stimulates proliferation through both protein kinase A (PKA)-dependent and PKA-independent signaling pathways and that phosphatidylinositol 3-kinase (PI3K) is required for cAMP-stimulated mitogenesis. In cells where cAMP is a mitogen, cAMP-elevating agents stimulate membrane ruffling, Akt phosphorylation, and p70 ribosomal S6 protein kinase (p70s6k) activity. cAMP effects on ruffle formation and Akt were PKA independent but sensitive to wortmannin. In contrast, cAMP-stimulated p70s6k activity was repressed by PKA inhibitors but not by wortmannin or microinjection of the N-terminal SH2 domain of the p85 regulatory subunit of PI3K, indicating that p70s6k and Akt can be regulated independently. Microinjection of highly specific inhibitors of PI3K or Rac1, or treatment with the p70s6k inhibitor rapamycin, impaired cAMP-stimulated DNA synthesis, demonstrating that PKA-dependent and -independent pathways contribute to cAMP-mediated mitogenesis. Direct elevation of PI3K activity through microinjection of an antibody that stimulates PI3K activity or stable expression of membrane-localized p110 was sufficient to confer hormone-independent DNA synthesis when accompanied by elevations in p70s6k activity. These findings indicate that multiple pathways contribute to cAMP-stimulated mitogenesis, only some of which are PKA dependent. Furthermore, they demonstrate that the ability of cAMP to stimulate both p70s6k- and PI3K-dependent pathways is an important facet of cAMP-regulated cell cycle progression.
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PMID:Protein kinase A-dependent and -independent signaling pathways contribute to cyclic AMP-stimulated proliferation. 1045 35

The Drosophila insulin receptor (INR) homolog includes an extension of approximately 400 amino acids at the carboxyl-terminal end of its beta subunit containing several tyrosine-based motifs known to mediate interactions with signaling proteins. In order to explore the role of this extension in INR function, mammalian expression vectors encoding either the complete INR beta subunit (beta-Myc) or the INR beta subunit without the carboxyl-terminal extension (betaDelta) were constructed, and the membrane-bound beta subunits were expressed in 293 and Madin-Darby canine kidney cells in the absence of the ligand-binding alpha subunits. beta-Myc and betaDelta proteins were constitutively active tyrosine kinases of 180 and 102 kDa, respectively. INR beta-Myc co-immunoprecipitated a phosphoprotein of 170 kDa identified as insulin receptor substrate-1 (IRS-1), whereas INR betaDelta did not, suggesting that the site of interaction was within the carboxyl-terminal extension. IRS-1 was phosphorylated on tyrosine to a much greater extent in cells expressing INR beta-Myc than in parental or INR betaDelta cells. Despite this, a variety of PTB or SH2 domain-containing signaling proteins, including IRS-2, mSos-1, Shc, p85 subunit of phosphatidylinositol 3-kinase, SHP-2, Raf-1, and JAK2, were not associated with the INR beta-Myc.IRS-1 complex. Overexpression of INR beta-Myc and betaDelta kinases conferred an equivalent increase in cell proliferation in both 293 and Madin-Darby canine kidney cells, indicating that this growth response is independent of the carboxyl-terminal extension. However, INR beta-Myc-expressing cells exhibited enhanced survival relative to parental and betaDelta cells, suggesting that the carboxyl-terminal extension, through its interaction with IRS-1, plays a role in the regulation of cell death.
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PMID:The carboxyl terminal extension of the Drosophila insulin receptor homologue binds IRS-1 and influences cell survival. 1045 77

Phosphoinositide 3-Kinases (PI3-Kinases) are a family of dual specificity enzymes with a unique lipid kinase activity toward the D-3 position of the inositol ring of phosphoinositides and a less well characterized serine/threonine protein kinase activity. Class IA PI3-Kinases comprise a 110-120 kDa catalytic subunit (usually termed p110) and an 85 kDa or 50 to 55 kDa regulatory subunit (often called p85). cDNAs for three mammalian Class IA PI3-Kinase catalytic subunits designated p110alpha, p110beta, and p110delta have been cloned from several species. A YAC clone for the human p110alpha gene has also been cloned and mapped to chromosome 3q26.3. However, structural organization for any of the PI3-Kinase p110alpha genes has not been reported. Here, we report the cloning, structural organization, and chromosomal localization of the mouse PI3-Kinase p110alpha gene. The translated portion of the mouse p110alpha gene is encoded by 19 exons that span at least 24 kb. Dual color fluorescence in situ hybridization (FISH) was performed to determine the chromosomal localization of the mouse PI3-Kinase p110alpha gene. FISH results and DAPI banding demonstrated localization of the p110alpha gene to band B on mouse chromosome 3, a region syntenic with human chromosome 3q26.3.
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PMID:Mouse phosphoinositide 3-kinase p110alpha gene: cloning, structural organization, and localization to chromosome 3 band B. 1046 94


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