scholarly journals RHO-associated protein kinase alpha potentiates insulin-induced MAP kinase activation in Xenopus oocytes

1999 ◽  
Vol 112 (13) ◽  
pp. 2177-2184 ◽  
Author(s):  
N. Ohan ◽  
Y. Agazie ◽  
C. Cummings ◽  
R. Booth ◽  
M. Bayaa ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Map Kinase ◽  
Insulin Receptor ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Insulin Receptor Substrate ◽  
Germinal Vesicle Breakdown ◽  
Kinase Activation ◽  
Insulin Signal Transduction

We recently identified Xenopus Rho-associated protein kinase alpha (xROKalpha) as a Xenopus insulin receptor substrate-1 binding protein and demonstrated that the non-catalytic carboxyl terminus of xROKalpha binds Xenopus insulin receptor substrate-1 and blocks insulin-induced MAP kinase activation and germinal vesicle breakdown in Xenopus oocytes. In the current study we further examined the role of xROKalpha in insulin signal transduction in Xenopus oocytes. We demonstrate that injection of mRNA encoding the xROKalpha kinase domain or full length xROKalpha enhanced insulin-induced MAP kinase activation and germinal vesicle breakdown. In contrast, injection of a kinase-dead mutant of xROKalpha or pre-incubation of oocytes with an xROKalpha inhibitor significantly reduced insulin-induced MAP kinase activation. To further dissect the mechanism by which xROKalpha may participate in insulin signalling, we explored a potential function of xROKalpha in regulating cellular Ras function, since insulin-induced MAP kinase activation and germinal vesicle breakdown is known to be a Ras-dependent process. We demonstrate that whereas injection of mRNA encoding c-H-Ras alone induced xMAP kinase activation and GVBD in a very low percentage (about 10%) of injected oocytes, co-injection of mRNA encoding xROKalpha and c-H-Ras induced xMAP kinase activation and germinal vesicle breakdown in a significantly higher percentage (50-60%) of injected oocytes. These results suggest a novel function for xROKalpha in insulin signal transduction upstream of cellular Ras function.

Ha-rasVal-12,Thr-59 activates S6 kinase and p34cdc2 kinase in Xenopus oocytes: evidence for c-mosxe-dependent and -independent pathways

1990 ◽  
Vol 10 (1) ◽  
pp. 310-315
Author(s):  
C B Barrett ◽  
R M Schroetke ◽  
F A Van der Hoorn ◽  
S K Nordeen ◽  
J L Maller
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Antisense Oligonucleotides ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Protein Product ◽  
Germinal Vesicle Breakdown ◽  
S6 Kinase ◽  
Kinase Activation ◽  
Histone Kinase

Treatment with insulin or progesterone or microinjection of the transforming protein product of Ha-rasVal-12,Thr-59 (p21) is known to induce germinal vesicle breakdown in Xenopus oocytes. We have investigated the effect of p21 on S6 kinase and the H1 histone kinase of maturation-promoting factor in the presence and absence of antisense oligonucleotides against the c-mosxe proto-oncogene. Injection of p21 led to a rapid increase in S6 phosphorylation, with kinetics similar to those previously observed with insulin. Microinjection of c-mosxe antisense oligonucleotides inhibited germinal vesicle breakdown induced by p21 and totally abolished S6 kinase activation by insulin or progesterone but only partially inhibited activation by p21. However, the activation of p34cdc2 protein kinase by all three stimuli was blocked by antisense oligonucleotides. The results suggest that in oocyte maturation c-mosxe functions downstream of p21 but upstream of p34cdc2 and S6 kinase activation, although not all p21-induced events require c-mosxe.

scholarly journals Molecular cloning of an amphibian insulin receptor substrate 1-like cDNA and involvement of phosphatidylinositol 3-kinase in insulin-induced Xenopus oocyte maturation.

1995 ◽  
Vol 15 (7) ◽  
pp. 3563-3570 ◽  
Author(s):  
X J Liu ◽  
A Sorisky ◽  
L Zhu ◽  
T Pawson
Keyword(s):  
Insulin Receptor ◽  
Oocyte Maturation ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Insulin Receptor Substrate ◽  
Phosphorylation Sites ◽  
Glutathione S Transferase ◽  
Germinal Vesicle Breakdown

An insulin receptor substrate 1 (IRS-1)-like cDNA was isolated from a Xenopus ovary cDNA library by low-stringency hybridization using rat IRS-1 cDNA as a probe. The deduced amino acid sequence encoded by this cDNA (termed XIRS-L) is 67% identical (77% similar) to that of rat IRS-1. Significantly, all the insulin-induced tyrosine phosphorylation sites identified in rat IRS-1, including those responsible for binding to the Src homology domains of phosphatidylinositol (PI) 3-kinase, Syp and Grb2, are conserved in XIRS-L. Both mRNA and protein corresponding to the cloned XIRS-L can be detected in immature Xenopus oocytes. Recombinant XIRS-L protein produced in insect cells or a bacterial glutathione S-transferase fusion protein containing the putative PI 3-kinase binding site can be phosphorylated in vitro by purified insulin receptor kinase (IRK) domain, and the IRK-catalyzed phosphorylation renders both proteins capable of binding PI 3-kinase in Xenopus oocyte lysates. Another glutathione S-transferase fusion protein containing the C terminus of XIRS-L and including several putative tyrosine phosphorylation sites is also phosphorylated by IRK in vitro, but it failed to bind PI 3-kinase. Insulin stimulation of immature Xenopus oocytes activates PI 3-kinase in vivo [as indicated by an elevation of PI(3,4)P2 and PI(3,4,5)P3] as well as oocyte maturation (as indicated by germinal vesicle breakdown). Pretreatment of these oocytes with wortmannin inhibited insulin-induced activation of PI 3-kinase in vivo. The same treatment also abolished insulin-induced, but not progesterone-induced, germinal vesicle breakdown. These results (i) identify an IRS-1-like molecule in immature Xenopus oocytes, suggesting that the use of IRS-1-like Scr homology 2 domain-docking proteins in signal transduction is conserved in vertebrates, and (ii) strongly implicate PI 3-kinase as an essential effector of insulin-induced oocyte maturation.

scholarly journals Ha-rasVal-12,Thr-59 activates S6 kinase and p34cdc2 kinase in Xenopus oocytes: evidence for c-mosxe-dependent and -independent pathways.

1990 ◽  
Vol 10 (1) ◽  
pp. 310-315 ◽  
Author(s):  
C B Barrett ◽  
R M Schroetke ◽  
F A Van der Hoorn ◽  
S K Nordeen ◽  
J L Maller
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Antisense Oligonucleotides ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Protein Product ◽  
Germinal Vesicle Breakdown ◽  
S6 Kinase ◽  
Kinase Activation ◽  
Histone Kinase

Treatment with insulin or progesterone or microinjection of the transforming protein product of Ha-rasVal-12,Thr-59 (p21) is known to induce germinal vesicle breakdown in Xenopus oocytes. We have investigated the effect of p21 on S6 kinase and the H1 histone kinase of maturation-promoting factor in the presence and absence of antisense oligonucleotides against the c-mosxe proto-oncogene. Injection of p21 led to a rapid increase in S6 phosphorylation, with kinetics similar to those previously observed with insulin. Microinjection of c-mosxe antisense oligonucleotides inhibited germinal vesicle breakdown induced by p21 and totally abolished S6 kinase activation by insulin or progesterone but only partially inhibited activation by p21. However, the activation of p34cdc2 protein kinase by all three stimuli was blocked by antisense oligonucleotides. The results suggest that in oocyte maturation c-mosxe functions downstream of p21 but upstream of p34cdc2 and S6 kinase activation, although not all p21-induced events require c-mosxe.

scholarly journals Requirement of mosXe protein kinase for meiotic maturation of Xenopus oocytes induced by a cdc2 mutant lacking regulatory phosphorylation sites.

1992 ◽  
Vol 12 (7) ◽  
pp. 3192-3203 ◽  
Author(s):  
K M Pickham ◽  
A N Meyer ◽  
J Li ◽  
D J Donoghue
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Phosphorylation Sites ◽  
Germinal Vesicle Breakdown ◽  
Regulatory Phosphorylation

The p34cdc2 protein kinase is a component of maturation-promoting factor, the master regulator of the cell cycle in all eukaryotes. The activity of p34cdc2 is itself tightly regulated by phosphorylation and dephosphorylation. Predicted regulatory phosphorylation sites of Xenopus p34cdc2 were mutated in vitro, and in vitro-transcribed RNAs were injected into Xenopus oocytes. The cdc2 single mutants Thr-14----Ala and Tyr-15----Phe did not induce germinal vesicle breakdown (BVBD) upon microinjection into oocytes. In contrast, the cdc2 double mutant Ala-14/Phe-15 did induce GVBD. Both the Ala-14 and Ala-14/Phe-15p34cdc2 mutants were shown to coimmunoprecipitate cyclin B1 and to phosphorylate histone H1 in immune complex kinase assays. Microinjection of antisense oligonucleotides to c-mosXe was used to demonstrate the role of mos protein synthesis in the induction of GVBD by the Ala-14/Phe-15 cdc2 mutant. Thr-161 was also mutated. p34cdc2 single mutants Ala-161 and Glu-161 and triple mutants Ala-14/Phe-15/Ala-161 and Ala-14/Phe-15/Glu-161 failed to induce GVBD in oocytes and showed a decreased binding to cyclin B1 in coimmunoprecipitations. Each of the cdc2 mutants was also assayed by coinjection with cyclin B1 or c-mosXe RNA into oocytes. Several of the cdc2 mutants were found to affect the kinetics of cyclin B1 and/or mos-induced GVBD upon coinjection, although none affected the rate of progesterone-induced maturation. We demonstrate here the significance of Thr-14, Tyr-15, and Thr-161 of p34cdc2 in Xenopus oocyte maturation. In addition, these results suggest a regulatory role for mosXe in induction of oocyte maturation by the cdc2 mutant Ala-14/Phe-15.

scholarly journals Insulin signal transduction in rat small intestine: role of MAP kinases in expression of mucosal hydrolases

2001 ◽  
Vol 280 (2) ◽  
pp. G229-G240 ◽  
Author(s):  
Soheila Marandi ◽  
Nadine De Keyser ◽  
Alain Saliez ◽  
Anne-Sophie Maernoudt ◽  
Etienne Marc Sokal ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Insulin Receptor ◽  
Map Kinases ◽  
Kinase Inhibitor ◽  
Protein Kinase B ◽  
Insulin Signal Transduction ◽  
Mitogen Activated Protein ◽  
Src Homology ◽  
Mucosal Mass

The postreceptor events regulating the signal of insulin downstream in rat intestinal cells have not yet been analyzed. Our objectives were to identify the nature of receptor substrates and phosphorylated proteins involved in the signaling of insulin and to investigate the mechanism(s) by which insulin enhances intestinal hydrolases. In response to insulin, the following proteins were rapidly phosphorylated on tyrosine residues: 1) insulin receptor substrates-1 (IRS-1), -2, and -4; 2) phospholipase C-isoenzyme-γ; 3) the Ras-GTPase-activating protein (GAP) associated with Rho GAP and p62Src; 4) the insulin receptor β-subunit; 5) the p85 subunits of phosphatidylinositol 3-kinase (PI 3-kinase); 6) the Src homology 2 α-collagen protein; 7) protein kinase B; 8) mitogen-activated protein (MAP) kinase-1 and -2; and 9) growth receptor-bound protein-2. Compared with controls, insulin enhanced the intestinal activity of MAP kinase-2 and protein kinase B by two- and fivefold, respectively, but did not enhance p70/S6 ribosomal kinase. Administration of an antireceptor antibody or MAP-kinase inhibitor PD-98059 but not a PI 3-kinase inhibitor (wortmannin) to sucklings inhibited the effects of insulin on mucosal mass and enzyme expression. We conclude that normal rat enterocytes express all of the receptor substrates and mediators involved in different insulin signaling pathways and that receptor binding initiates a signal enhancing brush-border membrane hydrolase, which appears to be regulated by the cascade of MAP kinases but not by PI 3-kinase.

scholarly journals Raf-1 protein kinase is important for progesterone-induced Xenopus oocyte maturation and acts downstream of mos.

1993 ◽  
Vol 13 (7) ◽  
pp. 4197-4202 ◽  
Author(s):  
A J Muslin ◽  
A M MacNicol ◽  
L T Williams
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Oocyte Maturation ◽  
Sodium Dodecyl ◽  
Germinal Vesicle ◽  
Histone H1 ◽  
Dominant Negative ◽  
Xenopus Laevis Oocytes ◽  
Mobility Shift ◽  
Germinal Vesicle Breakdown

In somatic cells, the Raf-1 serine/threonine protein kinase is activated by several polypeptide growth factors. We investigated the role of Raf-1 in progesterone-induced meiotic maturation of Xenopus laevis oocytes. Raf-1 enzymatic activity and phosphorylation (reflected by a mobility shift on sodium dodecyl sulfate gels) were increased in oocytes following progesterone stimulation. The increase in Raf-1 activity was concurrent with an elevation in the activity of mitogen-activated protein (MAP) kinase. When RNA encoding an oncogenic form of Raf-1 (v-Raf) was injected into immature oocytes, MAP kinase mobility shift, germinal vesicle breakdown, and histone H1 phosphorylation increased markedly. When RNA encoding a dominant-negative version of Raf-1 was injected, progesterone-induced oocyte maturation was blocked. When RNA encoding Xenopus mos (mosxe) was injected into oocytes, Raf-1 and MAP kinase mobility shifts were observed after several hours. Also, when antisense mosxe oligonucleotides were injected into oocytes, progesterone-induced Raf-1 and MAP kinase mobility shifts were blocked. Finally, when antisense mosxe oligonucleotides were coinjected with v-Raf RNA into oocytes, histone H1 kinase activation, germinal vesicle breakdown, and MAP kinase mobility shift occurred. These findings suggest that Raf-1 activity is required for progesterone-induced oocyte maturation and that Raf-1 is downstream of mosxe activity.

Mitogen-activated protein kinase activity and microtuble organisation are altered by protein synthesis inhibition in maturing porcine oocytes

Zygote ◽  
1996 ◽  
Vol 4 (3) ◽  
pp. 191-198 ◽  
Author(s):  
Maki Inoue ◽  
Kunihiko Naito ◽  
Taisuke Nakayama ◽  
Eimei Sato
Keyword(s):  
Protein Synthesis ◽  
Map Kinase ◽  
Kinase Activity ◽  
Germinal Vesicle ◽  
Protein Kinase Activity ◽  
Protein Synthesis Inhibition ◽  
Germinal Vesicle Breakdown ◽  
Kinase Activation ◽  
Synthesis Inhibition ◽  
Mitogen Activated Protein

SummaryPreviously we have shown that mitogen-activated protein (MAP) kinase activity abruptly increases at the first metaphase (M1) and remains significantly higher than that at the germinal vesicle (GV) stage until the second metaphase (M2) in porcine oocytes cultured in vitro. The present paper describes how the mechanism of the blockage of meiotic maturation by protein sythesis inhibition involves MAP kinase regulation. Cycloheximide arrested both germinal vesicle breakdown (GVBD) and the normal transition from M1 to M2. MAP kinase activation was also reduced in these maturation-inhibited oocytes. By using immunofluorescence microscopy with the monoclonal antibody raised against rat α-tubulin, we showed that cycloheximide caused morphological abnormality in a spindle at M1, but not at M2. All these results indicate that in porcine oocytes: (1) GV blockage by protein synthesis inhibition involves the suppression of both histone H1 kinase and MAP kinase activation, (2) during the transition from M1 to M2, maintenance of a normal metaphasic spindle and high MAP kinase activity require protein synthesis, and (3) once the M2 cytoskeletal structures have been completed, and/or after the ‘critical period’, cytostatic factor activity is independent of protein synthesis.

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