scholarly journals Insulin-Activated Protein Kinase Cβ Bypasses Ras and Stimulates Mitogen-Activated Protein Kinase Activity and Cell Proliferation in Muscle Cells

2000 ◽  
Vol 20 (17) ◽  
pp. 6323-6333 ◽  
Author(s):  
Pietro Formisano ◽  
Francesco Oriente ◽  
Francesca Fiory ◽  
Matilde Caruso ◽  
Claudia Miele ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Antisense Oligonucleotide ◽  
Kinase Activity ◽  
Mitogen Activated Protein Kinase ◽  
Insulin Stimulation ◽  
Mapk Activity ◽  
Mitogen Activated Protein ◽  
Stimulation Of

ABSTRACT In L6 muscle cells expressing wild-type human insulin receptors (L6hIR), insulin induced protein kinase Cα (PKCα) and β activities. The expression of kinase-deficient IR mutants abolished insulin stimulation of these PKC isoforms, indicating that receptor kinase is necessary for PKC activation by insulin. In L6hIR cells, inhibition of insulin receptor substrate 1 (IRS-1) expression caused a 90% decrease in insulin-induced PKCα and -β activation and blocked insulin stimulation of mitogen-activated protein kinase (MAPK) and DNA synthesis. Blocking PKCβ with either antisense oligonucleotide or the specific inhibitor LY379196 decreased the effects of insulin on MAPK activity and DNA synthesis by >80% but did not affect epidermal growth factor (EGF)- and serum-stimulated mitogenesis. In contrast, blocking c-Ras with lovastatin or the use of the L61,S186 dominant negative Ras mutant inhibited insulin-stimulated MAPK activity and DNA synthesis by only about 30% but completely blocked the effect of EGF. PKCβ block did not affect Ras activity but almost completely inhibited insulin-induced Raf kinase activation and coprecipitation with PKCβ. Finally, blocking PKCα expression by antisense oligonucleotide constitutively increased MAPK activity and DNA synthesis, with little effect on their insulin sensitivity. We make the following conclusions. (i) The tyrosine kinase activity of the IR is necessary for insulin activation of PKCα and -β. (ii) IRS-1 phosphorylation is necessary for insulin activation of these PKCs in the L6 cells. (iii) In these cells, PKCβ plays a unique Ras-independent role in mediating insulin but not EGF or other growth factor mitogenic signals.

Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation

1994 ◽  
Vol 14 (7) ◽  
pp. 4902-4911
Author(s):  
B Cheatham ◽  
C J Vlahos ◽  
L Cheatham ◽  
L Wang ◽  
J Blenis ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dna Synthesis ◽  
Mitogen Activated Protein Kinase ◽  
Phosphatidylinositol 3 Kinase ◽  
Insulin Stimulation ◽  
S6 Kinase ◽  
Kinase Activation ◽  
Mitogen Activated Protein ◽  
Stimulation Of ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by insulin and a variety of growth factors, but its exact role in signal transduction remains unclear. We have used a novel, highly specific inhibitor of PT 3-kinase to dissect the role of this enzyme in insulin action. Treatment of intact 3T3-L1 adipocytes with LY294002 produced a dose-dependent inhibition of insulin-stimulated PI 3-kinase (50% inhibitory concentration, 6 microM) with > 95% reduction in the levels of phosphatidylinositol-3,4,5-trisphosphate without changes in the levels of phosphatidylinositol-4-monophosphate or its derivatives. In parallel, there was a complete inhibition of insulin-stimulated phosphorylation and activation of pp70 S6 kinase. Inhibition of PI 3-kinase also effectively blocked insulin- and serum-stimulated DNA synthesis and insulin-stimulated glucose uptake by inhibiting translocation of GLUT 4 glucose transporters to the plasma membrane. By contrast, LY294002 had no effect on insulin stimulation of mitogen-activated protein kinase or pp90 S6 kinase. Thus, activation of PI 3-kinase plays a critical role in mammalian cells and is required for activation of pp70 S6 kinase and DNA synthesis and certain forms of intracellular vesicular trafficking but not mitogen-activated protein kinase or pp90 S6 kinase activation. These data suggest that PI 3-kinase is not only an important component but also a point of divergence in the insulin signaling network.

ATP activates DNA synthesis by acting on P2X receptors in human osteoblast-like MG-63 cells

2000 ◽  
Vol 279 (2) ◽  
pp. C510-C519 ◽  
Author(s):  
Ei'Ichiro Nakamura ◽  
Yasuhito Uezono ◽  
Ken'Ichiro Narusawa ◽  
Izumi Shibuya ◽  
Yosuke Oishi ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Kinase Activity ◽  
Thymidine Incorporation ◽  
P2x Receptors ◽  
Mitogen Activated Protein Kinase ◽  
Factor I ◽  
Mitogen Activated Protein ◽  
Intracellular Ca

In human osteoblast-like MG-63 cells, extracellular ATP increased [3H]thymidine incorporation and cell proliferation and synergistically enhanced platelet-derived growth factor- or insulin-like growth factor I-induced [3H]thymidine incorporation. ATP-induced [3H]thymidine incorporation was mimicked by the nonhydrolyzable ATP analogs adenosine 5′- O-(3-thiotriphosphate) and adenosine 5′-adenylylimidodiphosphate and was inhibited by the P2 purinoceptor antagonist suramin, suggesting involvement of P2 purinoceptors. The P2Y receptor agonist UTP and UDP and a P2Y receptor antagonist reactive blue 2 did not affect [3H]thymidine incorporation, whereas the P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2′,4-disulfonic acid inhibited ATP-induced [3H]thymidine incorporation, suggesting that ATP-induced DNA synthesis was mediated by P2X receptors. RT-PCR analysis revealed that MG-63 cells expressed P2X4, P2X5, P2X6, and P2X7, but not P2X1, P2X2, and P2X3, receptors. In fura 2-loaded cells, not only ATP, but also UTP, increased intracellular Ca2+concentration, and inhibitors for several Ca2+-activated protein kinases had no effect on ATP-induced DNA synthesis, suggesting that an increase in intracellular Ca2+concentration is not indispensable for ATP-induced DNA synthesis. ATP increased mitogen-activated protein kinase activity in a Ca2+-independent manner and synergistically enhanced platelet-derived growth factor- or insulin-like growth factor I-induced kinase activity. Furthermore, the mitogen-activated protein kinase kinase inhibitor PD-98059 totally abolished ATP-induced DNA synthesis. We conclude that ATP increases DNA synthesis and enhances the proliferative effects of growth factors through P2X receptors by activating a mitogen-activated protein kinase pathway.

scholarly journals Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation.

1994 ◽  
Vol 14 (7) ◽  
pp. 4902-4911 ◽  
Author(s):  
B Cheatham ◽  
C J Vlahos ◽  
L Cheatham ◽  
L Wang ◽  
J Blenis ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dna Synthesis ◽  
Mitogen Activated Protein Kinase ◽  
Phosphatidylinositol 3 Kinase ◽  
Insulin Stimulation ◽  
S6 Kinase ◽  
Kinase Activation ◽  
Mitogen Activated Protein ◽  
Stimulation Of ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by insulin and a variety of growth factors, but its exact role in signal transduction remains unclear. We have used a novel, highly specific inhibitor of PT 3-kinase to dissect the role of this enzyme in insulin action. Treatment of intact 3T3-L1 adipocytes with LY294002 produced a dose-dependent inhibition of insulin-stimulated PI 3-kinase (50% inhibitory concentration, 6 microM) with > 95% reduction in the levels of phosphatidylinositol-3,4,5-trisphosphate without changes in the levels of phosphatidylinositol-4-monophosphate or its derivatives. In parallel, there was a complete inhibition of insulin-stimulated phosphorylation and activation of pp70 S6 kinase. Inhibition of PI 3-kinase also effectively blocked insulin- and serum-stimulated DNA synthesis and insulin-stimulated glucose uptake by inhibiting translocation of GLUT 4 glucose transporters to the plasma membrane. By contrast, LY294002 had no effect on insulin stimulation of mitogen-activated protein kinase or pp90 S6 kinase. Thus, activation of PI 3-kinase plays a critical role in mammalian cells and is required for activation of pp70 S6 kinase and DNA synthesis and certain forms of intracellular vesicular trafficking but not mitogen-activated protein kinase or pp90 S6 kinase activation. These data suggest that PI 3-kinase is not only an important component but also a point of divergence in the insulin signaling network.

Role of the PI3K/PKB signaling pathway in cAMP-mediated translocation of rat liver Ntcp

1999 ◽  
Vol 277 (6) ◽  
pp. G1165-G1172 ◽  
Author(s):  
Cynthia R. L. Webster ◽  
M. Sawkat Anwer
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Signaling Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Phosphatidylinositol 3 Kinase ◽  
Pi3k Inhibitors ◽  
Mapk Activity ◽  
Mitogen Activated Protein ◽  
Pi3k Signaling Pathway ◽  
Stimulation Of

cAMP stimulates Na+-taurocholate (TC) cotransport by translocating the Na+-TC-cotransporting peptide (Ntcp) to the plasma membrane. The present study was undertaken to determine whether the phosphatidylinositol-3-kinase (PI3K)-signaling pathway is involved in cAMP-mediated translocation of Ntcp. The ability of cAMP to stimulate TC uptake declined significantly when hepatocytes were pretreated with PI3K inhibitors wortmannin or LY-294002. Wortmannin inhibited cAMP-mediated translocation of Ntcp to the plasma membrane. cAMP stimulated protein kinase B (PKB) activity by twofold within 5 min, an effect inhibited by wortmannin. Neither basal mitogen-activated protein kinase (MAPK) activity nor cAMP-mediated inhibition of MAPK activity was affected by wortmannin. cAMP also stimulated p70S6K activity. However, rapamycin, an inhibitor of p70S6K, failed to inhibit cAMP-mediated stimulation of TC uptake, indicating that the effect of cAMP is not mediated via p70S6K. Cytochalasin D, an inhibitor of actin filament formation, inhibited the ability of cAMP to stimulate TC uptake and Ntcp translocation. Together, these results suggest that the stimulation of TC uptake and Ntcp translocation by cAMP may be mediated via the PI3K/PKB signaling pathway and requires intact actin filaments.

333 DYNAMICS OF MITOGEN-ACTIVATED PROTEIN KINASE ACTIVITY IN PIG OOCYTES FOLLOWING PARTHENOGENETIC ACTIVATION BY DIFFERENT METHODS

2007 ◽  
Vol 19 (1) ◽  
pp. 282
Author(s):  
L. Nanassy ◽  
K. Lee ◽  
A. Javor ◽  
Z. Machaty
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Mitogen Activated Protein Kinase ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Mapk Activity ◽  
Mitogen Activated Protein ◽  
Pronuclear Formation

Cell cycle progression during mitosis and meiosis is known to be regulated by the M-phase promoting factor (MPF). However, recent findings revealed that mitogen-activated protein kinase (MAPK) also plays an important regulatory role during transition through the cell cycle. At fertilization the activity of MAPK drops shortly after MPF inactivation; the objective of this study was to investigate the dynamics of MAPK activity in pig oocytes after different activation methods. In vitro-matured oocytes were allocated to 3 groups. In group 1 (EP), the oocytes were activated by 2 DC pulses of 1.2 kV cm-1, 60 �s each. In the second group (EP + BU), the oocytes were electroporated and incubated for 4 h in 100 �M butyrolactone I (BU, an inhibitor of cdc2 kinase). In group 3 (EP + CHX), the oocytes were electroporated and treated for 5 h with 10 �g mL-1 cycloheximide (CHX, a protein synthesis inhibitor). After electroporation all oocytes were incubated in 7.5 �g mL-1 cytochalasin B for 4 h. Some oocytes were used to determine MAPK activity at 0, 1, 2, 3, 4, 5, and 6 h after electroporation using a MAPK assay kit. The assay measures MAPK activity by determining the phosphorylation of myelin basic protein by MAPK using the transfer of the γ-phosphate of [γ-32P] ATP. Pronuclear formation was evaluated at 6 h after electroporation; blastocyst formation and total cell numbers per embryo were determined after a 7-day culture in PZM-3 medium. Pronuclear formation was compared by the chi-square test, blastocyst formation was assessed using ANOVA, and the kinase activity was evaluated using the Student t-test. Pronuclear formation was highest in the combined methods [69.39% (EP) vs. 86.32% (EP + BU) and 87.56 % (EP + CHX); P < 0.05]. Similarly, the combined methods supported better development to the blastocyst stage [25.06 � 7.96% (EP), 58.32 � 7.62% (EP + BU), and 63.91 � 6.35% (EP + CHX); P < 0.05], whereas the average cell numbers of the blastocysts did not differ (47.11 � 3.12, 46.56 � 2.33, and 44.04 � 1.86, respectively). The initial MAPK activity was 0.123 � 0.017 pmol/min/oocyte which, after 1 h, dropped in all cases to values of 0.069 � 0.009 (EP), 0.072 � 0.007 (EP + BU), and 0.077 � 0.012 (EP + CHX) pmol/min/oocyte (P < 0.05). The MAPK activity in the EP group reached its lowest level at 3 h (0.057 � 0.007 pmol/min/oocyte); however, at 4 h it started to recover and by 6 h the activity (0.079 � 0.022 pmol/min/oocyte) did not differ from that of the non-activated oocytes. In the other groups, MAPK activity stayed low, and by the end of the experimental period it was significantly lower than that in the nontreated metaphase II oocytes (P < 0.05). The results indicate that electroporation followed by protein kinase inhibition or protein synthesis inhibition leads to the efficient inactivation of MAPK activity, and confirm our earlier findings that these combined treatments support superior embryo development after oocyte activation.

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