343 PHOSPHATIDYLINOSITOL 3-KINASE IS INVOLVED IN THE MAINTENANCE OF METAPHASE II ARREST IN PORCINE OOCYTES MATURED IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 328
Author(s):  
N. Kashiwazaki ◽  
M. Shimada ◽  
J. Ito
Keyword(s):  
Protein Kinase ◽  
Time Dependent ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Mapk Activity ◽  
Pronuclear Formation ◽  
Pkb Phosphorylation ◽  
Phosphatidylinositol 3

It has been reported that phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB) pathway plays a crucial role in the meiotic resumption and progression to the metaphase II (MII) stage of oocytes. However, the role of this pathway in meiotic arrest at the MII stage (cytostatic activity) is not well understood. In this study, the effect of a PI3K inhibitor, LY294002, on the mitogen-activated protein kinase (MAPK) and p34cdc2 kinase activities of matured porcine oocytes was examined. Immature oocytes were collected from ovaries and cultured in modified NCSU37 up to 48 hr. After culture, cumulus cells were removed and oocytes were cultured up to 24 h in medium supplemented with 25 or 50 μM LY294002. Groups of 10 or 20 oocytes were collected at each culture period for in vitro kinase assay of p34cdc2 kinase and MAPK, respectively. Groups of 40 oocytes were also used for detection of PKB phosphorylation by Western blotting. After maturation culture, both the p34cdc2 kinase and MAPK activities in the oocytes were gradually decreased in a time-dependent manner. Although 25 μM LY294002 did not affect either the p34cdc2 kinase or MAPK activities, 50 μM LY294002 suppressed the PKB phosphorylation and slightly decreased MAPK activity, but not the p34cdc2 kinase activity. Next, the effect of 10 μM Ca2+ ionophore which was reported as inducing a transient decrease of p342+ kinase but not MAPK activities, was examined in LY294002-treated oocytes. Pronuclear formation of the oocytes was also evaluated by the aceto-orcein staining. By additional treatment with LY294002 after Ca2+ ionophore, both the MAPK and p34cdc2 kinase activities were decreased in a time-dependent manner, concomitantly with improvement of pronuclear formation. Therefore, we concluded that PI3K is possibly involved in the maintenance of MAPK activity in matured porcine oocytes. The work was supported in part by Grant-in-Aid for Scientific Research from JSPS (KAKENHI) (21789253) to J.I. This work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan through a Grant-in-Aid for Matching Fund Subsidy for Private Universities to J.I. and N.K.

scholarly journals Phosphatidylinositol 3-Kinase-mediated Endocytosis of Renal Na+,K+-ATPase α Subunit in Response to Dopamine

1998 ◽  
Vol 9 (5) ◽  
pp. 1209-1220 ◽  
Author(s):  
Alexander V. Chibalin ◽  
Juleen R. Zierath ◽  
Adrian I. Katz ◽  
Per-Olof Berggren ◽  
Alejandro M. Bertorello
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Α Subunit ◽  
Kinase C ◽  
Dose Dependent ◽  
Α Subunits ◽  
K Activity ◽  
Phosphatidylinositol 3

Dopamine (DA) inhibition of Na+,K+-ATPase in proximal tubule cells is associated with increased endocytosis of its α and β subunits into early and late endosomes via a clathrin vesicle-dependent pathway. In this report we evaluated intracellular signals that could trigger this mechanism, specifically the role of phosphatidylinositol 3-kinase (PI 3-K), the activation of which initiates vesicular trafficking and targeting of proteins to specific cell compartments. DA stimulated PI 3-K activity in a time- and dose-dependent manner, and this effect was markedly blunted by wortmannin and LY 294002. Endocytosis of the Na+,K+-ATPase α subunit in response to DA was also inhibited in dose-dependent manner by wortmannin and LY 294002. Activation of PI 3-K generally occurs by association with tyrosine kinase receptors. However, in this study immunoprecipitation with a phosphotyrosine antibody did not reveal PI 3-K activity. DA-stimulated endocytosis of Na+,K+-ATPase α subunits required protein kinase C, and the ability of DA to stimulate PI 3-K was blocked by specific protein kinase C inhibitors. Activation of PI 3-K is mediated via the D1 receptor subtype and the sequential activation of phospholipase A2, arachidonic acid, and protein kinase C. The results indicate a key role for activation of PI 3-K in the endocytic sequence that leads to internalization of Na+,K+-ATPase α subunits in response to DA, and suggest a mechanism for the participation of protein kinase C in this process.

scholarly journals An Intramolecular Association between Two Domains of the Protein Kinase Fused Is Necessary for Hedgehog Signaling

2004 ◽  
Vol 24 (23) ◽  
pp. 10397-10405 ◽  
Author(s):  
Manuel Ascano ◽  
David J. Robbins
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Hedgehog Signaling ◽  
Membrane Vesicles ◽  
Kinase Domain ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Intramolecular Association

ABSTRACT The protein kinase Fused (Fu) is an integral member of the Hedgehog (Hh) signaling pathway. Although genetic studies demonstrate that Fu is required for the regulation of the Hh pathway, the mechanistic role that it plays remains largely unknown. Given our difficulty in developing an in vitro kinase assay for Fu, we reasoned that the catalytic activity of Fu might be highly regulated. Several mechanisms are known to regulate protein kinases, including self-association in either an intra- or an intermolecular fashion. Here, we provide evidence that Hh regulates Fu through intramolecular association between its kinase domain (ΔFu) and its carboxyl-terminal domain (Fu-tail). We show that ΔFu and Fu-tail can interact in trans, with or without the kinesin-related protein Costal 2 (Cos2). However, since the majority of Fu is found associated with Cos2 in vivo, we hypothesized that Fu-tail, which binds Cos2 directly, would be able to tether ΔFu to Cos2. We demonstrate that ΔFu colocalizes with Cos2 in the presence of Fu-tail and that this colocalization occurs on a subset of membrane vesicles previously characterized to be important for Hh signal transduction. Additionally, expression of Fu-tail in fu mutant flies that normally express only the kinase domain rescues the fu wing phenotype. Therefore, reestablishing the association between these two domains of Fu in trans is sufficient to restore Hh signal transduction in vivo. In such a manner we validate our hypothesis, demonstrating that Fu self-associates and is functional in an Hh-dependent manner. Our results here enhance our understanding of one of the least characterized, yet critical, components of Hh signal transduction.

scholarly journals Adrenocorticotrophic hormone stimulates phosphotyrosine phosphatase SHP2 in bovine adrenocortical cells: phosphorylation and activation by cAMP-dependent protein kinase

2000 ◽  
Vol 352 (2) ◽  
pp. 483-490 ◽  
Author(s):  
Stéphane ROCCHI ◽  
Isabelle GAILLARD ◽  
Emmanuel VAN OBBERGHEN ◽  
Edmond M. CHAMBAZ ◽  
Isabelle VILGRAIN
Keyword(s):  
Protein Kinase ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Adrenocorticotrophic Hormone ◽  
Adrenocortical Cells ◽  
Dependent Protein Kinase ◽  
Phosphotyrosine Phosphatase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

During activation of adrenocortical cells by adrenocorticotrophic hormone (ACTH), tyrosine dephosphorylation of paxillin is stimulated and this correlates with protrusion of filopodial structures and a decreased number of focal adhesions. These effects are inhibited by Na3VO4, a phosphotyrosine phosphatase inhibitor [Vilgrain, Chinn, Gaillard, Chambaz and Feige (1998) Biochem. J. 332, 533–540]. However, the tyrosine phosphatases involved in these processes remain to be identified. In this study, we provide evidence that the Src homology domain (SH)2-containing phosphotyrosine phosphatase (SHP)2, but not SHP1, is expressed in adrenocortical cells and is phosphorylated upon ACTH challenge. ACTH (10-8M) treatment of 32P-labelled adrenocortical cells resulted in an increase in phosphorylated SHP2. By probing SHP2-containing immunoprecipitates with an antibody to phosphoserine we found that SHP2 was phosphorylated on serine in ACTH-treated cells in a dose- and time-dependent manner. Furthermore, using an in vitro kinase assay, we showed that SHP2 was a target for cAMP-dependent protein kinase (PKA). Serine was identified as the only target amino acid phosphorylated in SHP2. Phosphorylation of SHP2 by PKA resulted in a dramatic stimulation of phosphatase activity measured either with insulin receptor substrate-1 or with the synthetic peptide [32P]poly(Glu/Tyr) as substrate. In an in-gel assay of SHP2-containing immunoprecipitates, phosphorylated in vitro by PKA or isolated from adrenocortical cells treated with 10nM ACTH, a pronounced activation of SHP2 activity was shown. These observations clearly support the idea that a PKA-mediated signal transduction pathway contributes to SHP2 regulation in adrenocortical cells and point to SHP2 as a possible mediator of the effects of ACTH.

scholarly journals Phosphatidylinositol 3-Kinase/Protein Kinase Cζ-Induced Phosphorylation of Sp1 and p107 Repressor Release Have a Critical Role in Histone Deacetylase Inhibitor-Mediated Depression of Transcription of the Luteinizing Hormone Receptor Gene

2006 ◽  
Vol 26 (18) ◽  
pp. 6748-6761 ◽  
Author(s):  
Ying Zhang ◽  
Mingjuan Liao ◽  
Maria L. Dufau
Keyword(s):  
Protein Kinase ◽  
Luteinizing Hormone ◽  
Histone Deacetylase ◽  
Hormone Receptor ◽  
Gene Activation ◽  
Gene Promoter ◽  
Luteinizing Hormone Receptor ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

ABSTRACT We have demonstrated that silencing of luteinizing hormone receptor (LHR) gene transcription is mediated via a proximal Sp1 site at its promoter. Trichostatin A (TSA) induced histone acetylation and gene activation in JAR cells that prevailed in the absence of changes in Sp1/Sp3 expression, their binding activity, disassociation of the histone deacetylase/mSin3A complex from the Sp1 site, or demethylation of the promoter. This indicated a different mechanism involved in TSA-induced derepression. The present studies have revealed that phosphatidylinositol 3-kinase/protein kinase Cζ (PI3K/PKCζ)-mediated Sp1 phosphorylation accounts for Sp1 site-dependent LHR gene activation. TSA caused marked phosphorylation of Sp1 at serine 641 in JAR and MCF-7 cells. Blockade of PI3K or PKCζ activity by specific inhibitors, kinase-deficient mutants, or small interfering RNA abolished the effect of TSA on the LHR gene and Sp1 phosphorylation. PKCζ was shown to associate with Sp1, and this association was enhanced by TSA. Sp1 phosphorylation at serine 641 was required for the release of the pRb homologue p107 from the LHR gene promoter, while p107 acted as a repressor of the LHR gene. Inhibition of PKCζ activity blocked the dissociation of p107 from the LHR gene promoter and markedly reduced Sp1 phosphorylation and transcription. These results have demonstrated that phosphorylation of Sp1 by PI3K/PKCζ is critical for TSA-activated LHR gene expression. These studies have revealed a novel mechanism of TSA action through derecruitment of a repressor from the LHR gene promoter in a PI3K/PKCζ-induced Sp1 phosphorylation-dependent manner.

scholarly journals Phosphatidylinositol 3-kinase activation is required for insulin-stimulated sodium transport in A6 cells

1998 ◽  
Vol 274 (4) ◽  
pp. E611-E617 ◽  
Author(s):  
Rae D. Record ◽  
Larry L. Froelich ◽  
Chris J. Vlahos ◽  
Bonnie L. Blazer-Yost
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Sodium Transport ◽  
Specific Inhibitor ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Insulin Receptor Tyrosine Kinase ◽  
A6 Cells ◽  
Phosphatidylinositol 3

Insulin stimulates amiloride-sensitive sodium transport in models of the distal nephron. Here we demonstrate that, in the A6 cell line, this action is mediated by the insulin receptor tyrosine kinase and that activation of phosphatidylinositol 3-kinase (PI 3-kinase) lies downstream of the receptor tyrosine kinase. Functionally, a specific inhibitor of PI 3-kinase, LY-294002, blocks basal as well as insulin-stimulated sodium transport in a dose-dependent manner (IC50 ≈ 6 μM). Biochemically, PI 3-kinase is present in A6 cells and is inhibited both in vivo and in vitro by LY-294002. Furthermore, a subsequent potential downstream signaling element, pp70 S6 kinase, is activated in response to insulin but does not appear to be part of the pathway involved in insulin-stimulated sodium transport. Together with previous reports, these results suggest that insulin may induce the exocytotic insertion of sodium channels into the apical membrane of A6 cells in a PI 3-kinase-mediated manner.

scholarly journals Relative importance of phosphatidylinositol-3 kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK3/1) signaling during maturational steroid-induced meiotic G2–M1 transition in zebrafish oocytes

Zygote ◽  
2017 ◽  
Vol 26 (1) ◽  
pp. 62-75 ◽  
Author(s):  
Debabrata Das ◽  
Poulomi Nath ◽  
Soumojit Pal ◽  
Sudip Hajra ◽  
Pritha Ghosh ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Relative Importance ◽  
Phosphatidylinositol 3 Kinase ◽  
Kinase Activation ◽  
Erk Activation ◽  
Mapk Signalling ◽  
Mitogen Activated Protein ◽  
Phosphatidylinositol 3

SummaryParticipation and relative importance of phosphatidylinositol-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling, either alone or in combination, have been investigated during 17α,20β-dihydroxy-4-pregnen-3-one (DHP)-induced meiotic G2−M1 transition in denuded zebrafish oocyte. Results demonstrate that concomitant with rapid phosphorylation (activation) of Akt (Ser473) and MAPK (ERK1/2) at as early as 15 min of incubation, DHP stimulation promotes enhanced an GVBD response and histone H1 kinase activation between 1 and 5 h in full-grown oocytes in vitro. While p-Akt reaches its peak at 60 to 90 min and undergoes downregulation to the basal level by 240 min, ERK1/2 phosphorylation (activation) increases gradually until 120 min and remains high thereafter. Although, priming with MEK1/2 inhibitor U0126 is without effect, PI3K inhibitors, wortmannin or LY294002, delay the GVBD response significantly (P < 0.001) until 3 h but not at 5 h of incubation. Interestingly, blocking PI3K and MEK function together could abrogate steroid-induced oocyte maturation at all time points tested. While DHP stimulation promotes phospho-PKA catalytic (p-PKAc) dephosphorylation (inactivation) between 30–120 min of incubation, simultaneous inhibition of PI3K and MEK1/2 kinases abrogates DHP action. Conversely, elevated intra-oocyte cAMP, through priming with either adenylyl cyclase (AC) activator forskolin (FK) or dibutyryl cAMP (db-cAMP), abrogates steroid-induced Akt and ERK1/2 phosphorylation. Taken together, these results suggest that DHP-induced Akt and ERK activation precedes the onset of meiosis (GVBD response) in a cAMP-sensitive manner and PI3K/Akt and MEK/MAPK pathways together have a pivotal influence in the downregulation of PKA and resumption of meiotic maturation in zebrafish oocytes in vitro.

A member of Forkhead family transcription factor, FKHRL1, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt activation pathway in erythropoietin signal transduction

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 941-949 ◽  
Author(s):  
Yoshifumi Kashii ◽  
Mie Uchida ◽  
Keita Kirito ◽  
Masaru Tanaka ◽  
Kousuke Nishijima ◽  
...  
Keyword(s):  
Kinase Assay ◽  
Leukemia Cell Line ◽  
Human Leukemia ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Erythroid Progenitor ◽  
Akt Activation ◽  
Activation Pathway ◽  
Pi3k Activity ◽  
Phosphatidylinositol 3

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is important for the regulation of a number of cellular responses. Serine/threonine kinase Akt (protein kinase B; PKB) is downstream of PI3K and activated by growth factors. This study found that erythropoietin (EPO) induced tyrosine phosphorylation of Akt in a time- and dose-dependent manner in EPO-dependent human leukemia cell line UT-7/EPO. In vitro kinase assay using histone H2B and glucose synthase kinase as substrates demonstrated that Akt was actually activated by EPO. EPO-induced phosphorylation of Akt was completely blocked by a PI3K-specific inhibitor, LY294002, at 10 μmol/L, indicating that activation of Akt by EPO is dependent on PI3K activity. In addition, overexpression of the constitutively active form of Akt on UT-7/EPO cells partially blocked apoptosis induced by withdrawal of EPO from the culture medium. This finding suggested that the PI3K-Akt activation pathway plays some role in the antiapoptotic effect of EPO. EPO induced phosphorylation of a member of the trancription factor Forkhead family, FKHRL1, at threonine 32 and serine 253 in a dose- and time-dependent manner in UT-7/EPO cells. Moreover, results showed that Akt kinase activated by EPO directly phosphorylated FKHRL1 protein and that FKHRL1 phosphorylation was completely dependent on PI3K activity as is the case for Akt. In conjunction with the evidence that FKHRL1 is expressed in normal human erythroid progenitor cells and erythroblasts, the results suggest that FKHRL1 plays an important role in erythropoiesis as one of the downstream target molecules of PI3K-Akt.

A member of Forkhead family transcription factor, FKHRL1, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt activation pathway in erythropoietin signal transduction

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 941-949 ◽  
Author(s):  
Yoshifumi Kashii ◽  
Mie Uchida ◽  
Keita Kirito ◽  
Masaru Tanaka ◽  
Kousuke Nishijima ◽  
...  
Keyword(s):  
Kinase Assay ◽  
Leukemia Cell Line ◽  
Human Leukemia ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Erythroid Progenitor ◽  
Akt Activation ◽  
Activation Pathway ◽  
Pi3k Activity ◽  
Phosphatidylinositol 3

Abstract The phosphatidylinositol 3-kinase (PI3K) signaling pathway is important for the regulation of a number of cellular responses. Serine/threonine kinase Akt (protein kinase B; PKB) is downstream of PI3K and activated by growth factors. This study found that erythropoietin (EPO) induced tyrosine phosphorylation of Akt in a time- and dose-dependent manner in EPO-dependent human leukemia cell line UT-7/EPO. In vitro kinase assay using histone H2B and glucose synthase kinase as substrates demonstrated that Akt was actually activated by EPO. EPO-induced phosphorylation of Akt was completely blocked by a PI3K-specific inhibitor, LY294002, at 10 μmol/L, indicating that activation of Akt by EPO is dependent on PI3K activity. In addition, overexpression of the constitutively active form of Akt on UT-7/EPO cells partially blocked apoptosis induced by withdrawal of EPO from the culture medium. This finding suggested that the PI3K-Akt activation pathway plays some role in the antiapoptotic effect of EPO. EPO induced phosphorylation of a member of the trancription factor Forkhead family, FKHRL1, at threonine 32 and serine 253 in a dose- and time-dependent manner in UT-7/EPO cells. Moreover, results showed that Akt kinase activated by EPO directly phosphorylated FKHRL1 protein and that FKHRL1 phosphorylation was completely dependent on PI3K activity as is the case for Akt. In conjunction with the evidence that FKHRL1 is expressed in normal human erythroid progenitor cells and erythroblasts, the results suggest that FKHRL1 plays an important role in erythropoiesis as one of the downstream target molecules of PI3K-Akt.

Sign in / Sign up

Export Citation Format

Share Document