Src family kinases are involved in the meiotic maturation of porcine oocytes

2015 ◽  
Vol 27 (7) ◽  
pp. 1097 ◽  
Author(s):  
Kateřina Kheilová ◽  
Jaroslav Petr ◽  
Tereza Žalmanová ◽  
Veronika Kučerová-Chrpová ◽  
Dalibor Řehák
Keyword(s):  
Fluorescence Intensity ◽  
Tyrosine Kinases ◽  
Germinal Vesicle ◽  
Active Role ◽  
Src Family Kinases ◽  
Mitogen Activated Protein Kinase ◽  
Meiotic Maturation ◽  
Mapk Activity ◽  
Mitogen Activated Protein

Mammalian meiotic maturation is regulated by changes in the phosphorylation state of proteins involved in signalling pathways. The regulatory proteins include the family of Src tyrosine kinases. Src family kinases (SFKs) are required for meiotic maturation of mouse oocytes, and it remains to be elucidated whether they play the same role in porcine oocytes. To clarify the role of SFKs in the meiotic maturation of porcine oocytes we used inhibition of SFKs, western blotting and immunolocalisation to determine the presence of SFKs and localisation in the oocytes and assays to determine the activity of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Inhibition of SFKs resulted in the disruption of oocyte maturation and led to a decline in MPF and MAPK activity. The fluorescence intensity of SFKs in the cytoplasm and membrane of MI oocytes decreased significantly compared with germinal vesicle oocytes. The highest fluorescence intensity for SFKs was detected on the membrane of MII oocytes. Only weak fluorescence was detected in the perichromosomal area of MI and MII oocytes. These results prove that SFKs play an active role in the meiotic maturation of porcine oocytes by regulating MPF and MAPK activity.

scholarly journals The Thrombopoietin Receptor Can Mediate Proliferation without Activation of the Jak-STAT Pathway

1997 ◽  
Vol 186 (12) ◽  
pp. 1947-1955 ◽  
Author(s):  
Marion Dorsch ◽  
Pang-Dian Fan ◽  
Nika N. Danial ◽  
Paul B. Rothman ◽  
Stephen P. Goff
Keyword(s):  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Signal Transducers ◽  
Thrombopoietin Receptor ◽  
Mitogen Activated Protein ◽  
Mutant Receptors ◽  
Nonreceptor Tyrosine Kinases ◽  
Stat Pathway

Cytokine receptors of the hematopoietic receptor superfamily lack intrinsic tyrosine kinase domains for the intracellular transmission of their signals. Instead all members of this family associate with Jak family nonreceptor tyrosine kinases. Upon ligand stimulation of the receptors, Jaks are activated to phosphorylate target substrates. These include STAT (signal transducers and activators of transcription) proteins, which after phosphorylation translocate to the nucleus and modulate gene expression. The exact role of the Jak-STAT pathway in conveying growth and differentiation signals remains unclear. Here we describe a deletion mutant of the thrombopoietin receptor (c-mpl) that has completely lost the capacity to activate Jaks and STATs but retains its ability to induce proliferation. This mutant still mediates TPO-induced phosphorylation of Shc, Vav, mitogen-activated protein kinase (MAPK) and Raf-1 as well as induction of c-fos and c-myc, although at somewhat reduced levels. Furthermore, we show that both wild-type and mutant receptors activate phosphatidylinositol (PI) 3-kinase upon thrombopoietin stimulation and that thrombopoietin-induced proliferation is inhibited in the presence of the PI 3-kinase inhibitor wortmannin. These results demonstrate that the Jak-STAT pathway is dispensable for the generation of mitogenic signals by a cytokine receptor.

scholarly journals SHP2 Nuclear/Cytoplasmic Trafficking in Granulosa Cells Is Essential for Oocyte Meiotic Resumption and Maturation

2021 ◽  
Vol 8 ◽  
Author(s):  
Muhammad Idrees ◽  
Vikas Kumar ◽  
Myeong-Don Joo ◽  
Niaz Ali ◽  
Keun-Woo Lee ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Germinal Vesicle ◽  
G Protein Coupled Receptors ◽  
Meiotic Maturation ◽  
Preimplantation Embryo Development ◽  
Src Homology 2 ◽  
Src Homology ◽  
A Site ◽  
G Protein Coupled

Src-homology-2-containing phosphotyrosine phosphatase (SHP2), a classic cytoplasmic protein and a major regulator of receptor tyrosine kinases and G protein-coupled receptors, plays a significant role in preimplantation embryo development. In this study, we deciphered the role of SHP2 in the somatic compartment of oocytes during meiotic maturation. SHP2 showed nuclear/cytoplasmic localization in bovine cumulus and human granulosa (COV434) cells. Follicle-stimulating hormone (FSH) treatment significantly enhanced cytoplasmic SHP2 localization, in contrast to the E2 treatment, which augmented nuclear localization. Enhanced cytoplasmic SHP2 was found to negatively regulate the expression of the ERα-transcribed NPPC and NPR2 mRNAs, which are vital for oocyte meiotic arrest. The co-immunoprecipitation results revealed the presence of the SHP2/ERα complex in the germinal vesicle-stage cumulus–oocyte complexes, and this complex significantly decreased with the progression of meiotic maturation. The complex formation between ERα and SHP2 was also confirmed by using a series of computational modeling methods. To verify the correlation between SHP2 and NPPC/NPR2, SHP2 was knocked down via RNA interference, and NPPC and NPR2 mRNAs were analyzed in the control, E2, and FSH-stimulated COV434 cells. Furthermore, phenyl hydrazonopyrazolone sulfonate 1, a site-directed inhibitor of active SHP2, showed no significant effect on the ERα-transcribed NPPC and NPR2 mRNAs. Taken together, these findings support a novel nuclear/cytoplasmic role of SHP2 in oocyte meiotic resumption and maturation.

Critical tyrosine residues regulate the enzymatic and biological activity of Raf-1 kinase

1993 ◽  
Vol 13 (11) ◽  
pp. 7170-7179 ◽  
Author(s):  
J R Fabian ◽  
I O Daar ◽  
D K Morrison
Keyword(s):  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Expression System ◽  
Mitogen Activated Protein Kinase ◽  
Single Amino Acid ◽  
Meiotic Maturation ◽  
Phosphorylation Sites ◽  
Serine Threonine Kinase ◽  
Mitogen Activated Protein

The serine/threonine kinase activity of the Raf-1 proto-oncogene product is stimulated by the activation of many tyrosine kinases, including growth factor receptors and pp60v-src. Recent studies of growth factor signal transduction pathways demonstrate that Raf-1 functions downstream of activated tyrosine kinases and p21ras and upstream of mitogen-activated protein kinase. However, coexpression of both activated tyrosine kinases and p21ras is required for maximal activation of Raf-1 in the baculovirus-Sf9 expression system. In this study, we investigated the role of tyrosine kinases and tyrosine phosphorylation in the regulation of Raf-1 activity. Using the baculovirus-Sf9 expression system, we identified Tyr-340 and Tyr-341 as the major tyrosine phosphorylation sites of Raf-1 when coexpressed with activated tyrosine kinases. Introduction of a negatively charged residue that may mimic the effect of phosphorylation at these sites activated the catalytic activity of Raf-1 and generated proteins that could transform BALB/3T3 cells and induce the meiotic maturation of Xenopus oocytes. In contrast, substitution of noncharged residues that were unable to be phosphorylated produced a protein that could not be enzymatically activated by tyrosine kinases and that could block the meiotic maturation of oocytes induced by components of the receptor tyrosine kinase pathway. These findings demonstrate that maturation of the tyrosine phosphorylation sites can dramatically alter the function of Raf-1. In addition, this is the first report that a transforming Raf-1 protein can be generated by a single amino acid substitution.

scholarly journals Critical tyrosine residues regulate the enzymatic and biological activity of Raf-1 kinase.

1993 ◽  
Vol 13 (11) ◽  
pp. 7170-7179 ◽  
Author(s):  
J R Fabian ◽  
I O Daar ◽  
D K Morrison
Keyword(s):  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Expression System ◽  
Mitogen Activated Protein Kinase ◽  
Single Amino Acid ◽  
Meiotic Maturation ◽  
Phosphorylation Sites ◽  
Serine Threonine Kinase ◽  
Mitogen Activated Protein

The serine/threonine kinase activity of the Raf-1 proto-oncogene product is stimulated by the activation of many tyrosine kinases, including growth factor receptors and pp60v-src. Recent studies of growth factor signal transduction pathways demonstrate that Raf-1 functions downstream of activated tyrosine kinases and p21ras and upstream of mitogen-activated protein kinase. However, coexpression of both activated tyrosine kinases and p21ras is required for maximal activation of Raf-1 in the baculovirus-Sf9 expression system. In this study, we investigated the role of tyrosine kinases and tyrosine phosphorylation in the regulation of Raf-1 activity. Using the baculovirus-Sf9 expression system, we identified Tyr-340 and Tyr-341 as the major tyrosine phosphorylation sites of Raf-1 when coexpressed with activated tyrosine kinases. Introduction of a negatively charged residue that may mimic the effect of phosphorylation at these sites activated the catalytic activity of Raf-1 and generated proteins that could transform BALB/3T3 cells and induce the meiotic maturation of Xenopus oocytes. In contrast, substitution of noncharged residues that were unable to be phosphorylated produced a protein that could not be enzymatically activated by tyrosine kinases and that could block the meiotic maturation of oocytes induced by components of the receptor tyrosine kinase pathway. These findings demonstrate that maturation of the tyrosine phosphorylation sites can dramatically alter the function of Raf-1. In addition, this is the first report that a transforming Raf-1 protein can be generated by a single amino acid substitution.

Role of cAMP inhibition of p44/p42 mitogen-activated protein kinase in potentiation of protein secretion in rat lacrimal gland

2007 ◽  
Vol 293 (5) ◽  
pp. C1551-C1560 ◽  
Author(s):  
Chika Funaki ◽  
Robin R. Hodges ◽  
Darlene A. Dartt
Keyword(s):  
Protein Kinase ◽  
Protein Secretion ◽  
Lacrimal Gland ◽  
Mitogen Activated Protein Kinase ◽  
Adrenergic Agonist ◽  
Mapk Activity ◽  
Mitogen Activated Protein ◽  
Epidermal Growth ◽  
Camp Levels

We previously found that addition of cAMP and a Ca2+/PKC-dependent agonist causes synergism or potentiation of protein secretion from rat lacrimal gland acini. In the present study we determined whether cAMP decreases p44/p42 mitogen-activated protein kinase (MAPK) activity in the lacrimal gland. Since we know that activation of MAPK attenuates protein secretion stimulated by Ca2+- and PKC-dependent agonists, we also determined whether this activation causes potentiation of secretion. Freshly prepared rat lacrimal gland acinar cells were incubated with dibutyryl cAMP (DBcAMP), carbachol (a cholinergic agonist), phenylephrine (an α1-adrenergic agonist), or epidermal growth factor (EGF). The latter three agonists are known to activate p44/p42 MAPK. p44/p42 MAPK activity and protein secretion were measured. As measured by Western blot analysis, DBcAMP inhibited both basal and agonist-stimulated p44/p42 MAPK activity. Cellular cAMP levels were increased by 1) using two different cell-permeant cAMP analogs, 2) activating adenylyl cyclase (L-858051), or 3) activation of Gs-coupled receptors (VIP). The cell-permeant cAMP analogs, L-858051, and VIP inhibited basal p44/p42 MAPK activity by 50, 40, and 40%, respectively. DBcAMP and VIP inhibited carbachol- and EGF-stimulated MAPK activity. cAMP, but not VIP, inhibited phenylephrine-stimulated MAPK activity. Potentiation of secretion was detected when carbachol, phenylephrine, or EGF was simultaneously added with DBcAMP. We conclude that increasing cellular cAMP levels inhibits p44/p42 MAPK activity and that this could account for potentiation of secretion obtained when cAMP was elevated and Ca2+ and PKC were increased by agonists.

scholarly journals Activation of p42 Mitogen-activated Protein Kinase (MAPK), but not c-Jun NH2-Terminal Kinase, Induces Phosphorylation and Stabilization of MAPK Phosphatase XCL100 inXenopus Oocytes

2002 ◽  
Vol 13 (2) ◽  
pp. 454-468 ◽  
Author(s):  
Michael L. Sohaskey ◽  
James E. Ferrell
Keyword(s):  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Meiotic Maturation ◽  
Dependent Manner ◽  
Limited Information ◽  
Mapk Activity ◽  
Dual Specificity ◽  
Mitogen Activated Protein ◽  
Mapk Phosphatase

Dual-specificity protein phosphatases are implicated in the direct down-regulation of mitogen-activated protein kinase (MAPK) activity in vivo. Accumulating evidence suggests that these phosphatases are components of negative feedback loops that restore MAPK activity to low levels after diverse physiological responses. Limited information exists, however, regarding their posttranscriptional regulation. We cloned two Xenopus homologs of the mammalian dual-specificity MAPK phosphatases MKP-1/CL100 and found that overexpression of XCL100 in G2-arrested oocytes delayed or prevented progesterone-induced meiotic maturation. Epitope-taggedXCL100 was phosphorylated on serine during G2 phase, and on serine and threonine in a p42 MAPK-dependent manner during M phase. Threonine phosphorylation mapped to a single residue, threonine 168. Phosphorylation of XCL100 had no measurable effect on its ability to dephosphorylate p42 MAPK. Similarly, mutation of threonine 168 to either valine or glutamate did not significantly alter the binding affinity of a catalytically inactive XCL100 protein for active p42 MAPK in vivo. XCL100 was a labile protein in G2-arrested and progesterone-stimulated oocytes; surprisingly, its degradation rate was increased more than twofold after exposure to hyperosmolar sorbitol. In sorbitol-treated oocytes expressing a conditionally active ΔRaf-DD:ER chimera, activation of the p42 MAPK cascade led to phosphorylation of XCL100 and a pronounced decrease in the rate of its degradation. Our results provide mechanistic insight into the regulation of a dual-specificity MAPK phosphatase during meiotic maturation and the adaptation to cellular stress.

scholarly journals Kinase Pathways in Chemoattractant-Induced Degranulation of Neutrophils: The Role of p38 Mitogen-Activated Protein Kinase Activated by Src Family Kinases

2000 ◽  
Vol 164 (8) ◽  
pp. 4321-4331 ◽  
Author(s):  
Attila Mócsai ◽  
Zoltán Jakus ◽  
Tibor Vántus ◽  
Giorgio Berton ◽  
Clifford A. Lowell ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Src Family Kinases ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein

scholarly journals Nucleus, Cytoskeleton, and Mitogen-Activated Protein Kinase p38 Dynamics during In Vitro Maturation of Porcine Oocytes

Animals ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 163
Author(s):  
Payungsuk Intawicha ◽  
Li-Kuang Tsai ◽  
Shih-Ying Yen ◽  
Neng-Wen Lo ◽  
Jyh-Cherng Ju
Keyword(s):  
Oocyte Maturation ◽  
Mammalian Cells ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Mitogen Activated Protein Kinase ◽  
Meiotic Maturation ◽  
Germinal Vesicle Breakdown ◽  
Mitogen Activated Protein ◽  
Porcine Oocyte

The mitogen-activated kinase (MAPK) p38, a member of the MAPK subfamily, is conserved in all mammalian cells and plays important roles in response to various physiologic cues, including mitogens and heat shock. In the present study, MAPK p38 protein expression in porcine oocytes was analyzed during in vitro maturation (IVM) by Western blotting and immunocytochemistry. The levels of p-p38 or activated p38 and p38 expression were at the lowest in the germinal vesicle (GV) stage oocyte, gradually rising at the germinal vesicle breakdown (GVBD) and then reaching a plateau throughout the IVM culture (p < 0.05). Similarly, the expression level of total p38 was also lower in the GV oocyte than in the oocyte of other meiotic stages and uprising after GVBD and remained high until the metaphase III (MII) stage (p < 0.05). In the GV stage, phosphorylated p38 (p-p38) was initially detectable in the ooplasm and subsequently became clear around the nucleus and localized in the ooplasm at GVBD (18 h post-culture). During the metaphase I (MI) and metaphase II (MII) stages, p-p38 was evenly distributed throughout the ooplasm after IVM for 30 or 42 h. We found that the subcellular localization increased in p-p38 expression throughout oocyte maturation (p < 0.05) and that dynamic reorganization of the cytoskeleton, including microfilaments and microtubules, was progressively changed during the course of meiotic maturation which was likely to be associated with the activation or networking of p38 with other proteins in supporting oocyte development. In conclusion, the alteration of p38 activation is essential for the regulation of porcine oocyte maturation, accompanied by the progressive reorganization and redistribution of the cytoskeleton and MAPK p38, respectively, in the ooplasm.

scholarly journals Activation of mitogen-activated protein kinase during meiotic maturation in porcine oocytes

Zygote ◽  
1995 ◽  
Vol 3 (3) ◽  
pp. 265-271 ◽  
Author(s):  
Maki Inoue ◽  
Kunihiko Naito ◽  
Fugaku Aoki ◽  
Yutaka Toyoda ◽  
Eimei Sato
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Kinase Activity ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Mitogen Activated Protein Kinase ◽  
Meiotic Maturation ◽  
Germinal Vesicle Breakdown ◽  
First Polar Body ◽  
Mitogen Activated Protein

SummaryTo investigate the involvement of mitogen-activated protein kinase(MAP kinase) in meiotic maturation of porcine oocytes, we assayed MAP kinase activity using basic protein(MBP) as a substrate. MAP kinase activity was low during the germinal vesicle stage, 0–20 h of culture. An abrupt increase was observed at metaphase I(30 h of culture), and activity remained significantly higher than that at 0 h until 50 h of culture, with a transient slight decrease at the time of first polar body extrusion (40 h). Detection of the kinase activity by an in-gel phosphorylation assay confirmed that the 42 and 44 kDa MAP kinases were significantly activated in 45 h cultured oocytes but not in 0 h oocytes, and just slightly in 20 h oocytes. In immunoblotting, however, the 42 and 44 kDa bands were detected in 0, 20 and 45 h cultured oocytes. Furthermore, the signal strength of the two bands did not change during the period of culture, but shifted up to 45 h, indicating that the activation of MAP kinase depended not on the synthesis but on the phosphorylation of this enzyme. These results suggest that the activation of MAP kinase is involved in the regulation of meiotic maturation of porcine oocytes, and especially in the regulation after germinal vesicle breakdown.

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