scholarly journals v-Src phosphorylation of connexin 43 on Tyr247 and Tyr265 disrupts gap junctional communication

2001 ◽  
Vol 154 (4) ◽  
pp. 815-828 ◽  
Author(s):  
Rui Lin ◽  
Bonnie J. Warn-Cramer ◽  
Wendy E. Kurata ◽  
Alan F. Lau
Keyword(s):  
Mammalian Cells ◽  
Connexin 43 ◽  
Experimental System ◽  
Mouse Cell ◽  
Mitogen Activated Protein Kinase ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Gap Junctional

The mechanism by which v-Src disrupts connexin (Cx)43 intercellular gap junctional communication (GJC) is not clear. In this study, we determined that Tyr247 (Y247) and the previously identified Tyr265 (Y265) site of Cx43 were the primary phosphorylation targets for activated Src in vitro. We established an in vivo experimental system by stably expressing v-Src and wild-type (wt) Cx43, or Y247F, Y265F, or Y247F/Y265F Cx43 mutants in a Cx43 knockout mouse cell line. Wt and mutant Cx43 localized to the plasma membrane in the absence or presence of v-Src. When coexpressed with v-Src, the Y247F, Y265F, and Y247F/Y265F Cx43 mutants exhibited significantly reduced levels of tyrosine phosphorylation compared with wt Cx43, indicating that Y247 and Y265 were phosphorylation targets of v-Src in vivo. Most importantly, GJC established by the Y247F, Y265F, and Y247F/Y265F Cx43 mutants was resistant to disruption by v-Src. Furthermore, we did not find evidence for a role for mitogen-activated protein kinase in mediating the disruption of GJC by v-Src. We conclude that phosphorylation on Y247 and Y265 of Cx43 is responsible for disrupting GJC in these mammalian cells expressing v-Src.

scholarly journals Interaction between Connexin50 and Mitogen-activated Protein Kinase Signaling in Lens Homeostasis

2009 ◽  
Vol 20 (10) ◽  
pp. 2582-2592 ◽  
Author(s):  
Teresa I. Shakespeare ◽  
Caterina Sellitto ◽  
Leping Li ◽  
Clio Rubinos ◽  
Xiaohua Gong ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Signal Transduction Pathways ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Mitogen Activated Protein ◽  
Gap Junctional

Both connexins and signal transduction pathways have been independently shown to play critical roles in lens homeostasis, but little is known about potential cooperation between these two intercellular communication systems. To investigate whether growth factor signaling and gap junctional communication interact during the development of lens homeostasis, we examined the effect of mitogen-activated protein kinase (MAPK) signaling on coupling mediated by specific lens connexins by using a combination of in vitro and in vivo assays. Activation of MAPK signaling pathways significantly increased coupling provided by Cx50, but not Cx46, in paired Xenopus laevis oocytes in vitro, as well as between freshly isolated lens cells in vivo. Constitutively active MAPK signaling caused macrophthalmia, cataract, glucose accumulation, vacuole formation in differentiating fibers, and lens rupture in vivo. The specific removal or replacement of Cx50, but not Cx46, ameliorated all five pathological conditions in transgenic mice. These results indicate that MAPK signaling specifically modulates coupling mediated by Cx50 and that gap junctional communication and signal transduction pathways may interact in osmotic regulation during postnatal fiber development.

scholarly journals Regulation of Gap Junctional Communication by Astrocytic Mitochondrial KATP Channels following Neurotoxin Administration in in vitro and in vivo Models

Neurosignals ◽  
2011 ◽  
Vol 19 (2) ◽  
pp. 63-74 ◽  
Author(s):  
Kewen Jiang ◽  
Jiangping Wang ◽  
Congying Zhao ◽  
Mei Feng ◽  
Zheng Shen ◽  
...  
Keyword(s):  
In Vivo Models ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Gap Junctional

scholarly journals Epidermal growth factor stimulates the disruption of gap junctional communication and connexin43 phosphorylation independent of 12-0-tetradecanoylphorbol 13-acetate-sensitive protein kinase C: the possible involvement of mitogen-activated protein kinase.

1993 ◽  
Vol 4 (8) ◽  
pp. 837-848 ◽  
Author(s):  
M Y Kanemitsu ◽  
A F Lau
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Mitogen Activated Protein ◽  
Epidermal Growth ◽  
Phosphopeptide Mapping ◽  
Gap Junctional

We previously reported that epidermal growth factor (EGF) induced the disruption of gap junctional communication (gjc) and serine phosphorylation of connexin43 (Cx43) in T51B rat liver epithelial cells. However, the cascade of events linking EGF receptor activation to these particular responses have not been fully characterized. Furthermore, the serine kinase(s) acting directly on Cx43 remain unidentified. In the current study, we demonstrate that downmodulation of 12-0-tetradecanoylphorbol 13-acetate (TPA)-sensitive protein kinase C (PKC) activity does not affect EGF's ability to reduce junctional permeability or phosphorylate Cx43 in T51B cells. EGF in the presence or absence of chronic TPA treatment stimulated marked increases in Cx43 phosphorylation on numerous sites as determined by two-dimensional tryptic phosphopeptide mapping. Computer-assisted sequence analysis of Cx43 identified several protein kinase phosphorylation consensus sites including two sites for mitogen-activated protein (MAP) kinase. EGF stimulated activation of MAP kinase in a time- and dose-dependent manner where the kinetics of kinase activity corroborated its possible involvement in mediating EGF's effects. Moreover, purified MAP kinase directly phosphorylated Cx43 on serine residues in vitro. Two-dimensional tryptic and chymotryptic phosphopeptide mapping demonstrated that the in vitro phosphopeptides represented a specific subset of the in vivo phosphopeptides produced in response to EGF after chronic TPA treatment. Therefore, EGF-induced disruption of gjc and phosphorylation of Cx43 may be mediated in part by MAP kinase in vivo.

scholarly journals Gap-junctional communication in mouse cumulus-oocyte complexes: implications for the mechanism of meiotic maturation

Reproduction ◽  
2002 ◽  
pp. 41-52 ◽  
Author(s):  
RJ Webb ◽  
H Bains ◽  
C Cruttwell ◽  
J Carroll
Keyword(s):  
Ovarian Function ◽  
Ovarian Follicle ◽  
Somatic Cells ◽  
Cumulus Cells ◽  
Meiotic Maturation ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Gap Junctional

The mechanisms underlying the hormonal stimulation of meiotic maturation are not understood. The most prevalent hypothesis is that hormone-induced maturation is stimulated by an increase in the intracellular messengers, cAMP or Ca2+. This study investigated whether Ca2+ transients in somatic cells can lead to Ca2+ transients in the oocyte, and whether hormones that stimulate meiotic maturation of mouse oocytes in vitro and in vivo stimulate an increase in intracellular Ca2+. Of a range of potential agonists of Ca2+ release, ATP and UTP were the only agents that stimulated Ca2+ release in cumulus cells. ATP-induced Ca2+ release is from intracellular stores, as the response is not blocked by chelation of extracellular Ca2+, but is inhibited by the Ca2+-ATPase inhibitor, thapsigargin. ATP and UTP are equipotent, consistent with the receptor being of the P2Y2 type. Confocal microscopy was used to show that ATP-induced Ca2+ release in cumulus cells leads to a Ca2+ increase in the oocyte. Inhibition of gap-junctional communication using carbenoxolone, as assayed by dye transfer, inhibited the diffusion of the Ca2+ signal from the cumulus cells to the oocyte. Thus, provided that a Ca2+ signal is generated in the somatic cells in response to maturation-inducing hormones, it is feasible that a Ca2+ transient is generated in the oocyte. However, FSH and EGF, both of which stimulate maturation in vitro, have no effect on Ca2+ in cumulus--oocyte complexes. Furthermore, LH, which leads to meiotic maturation in vivo, did not stimulate Ca2+ release in acutely isolated granulosa cells from preovulatory mouse follicles. These studies indicate that ATP may play a role in modulating ovarian function and that diffusion of Ca2+ signals through gap junctions may provide a means of communication between the somatic and germ cells of the ovarian follicle. However, our data are not consistent with a role for Ca2+-mediated communication in hormone-mediated induction of meiosis in mice.

scholarly journals Inflammatory conditions induce gap junctional communication between rat Kupffer cells both in vivo and in vitro

2007 ◽  
Vol 247 (2) ◽  
pp. 103-110 ◽  
Author(s):  
Eliseo A. Eugenín ◽  
Hernán E. González ◽  
Helmuth A. Sánchez ◽  
María C. Brañes ◽  
Juan C. Sáez
Keyword(s):  
Kupffer Cells ◽  
Gap Junctional Communication ◽  
Inflammatory Conditions ◽  
Junctional Communication ◽  
Gap Junctional

REGULATION OF CUMULUS-OOCYTE GAP-JUNCTIONAL COMMUNICATION IN PORCINE DURING IN VITRO MATURATION: GONADOTROPIN-INDEPENDENT INCREASES OF CONNEXIN 43 PROTEIN

2007 ◽  
Vol 77 (Suppl_1) ◽  
pp. 141-141
Author(s):  
Maxime Sasseville ◽  
Marie-Claude Gagnon ◽  
Christine Guillemette ◽  
Robert Gilchrist ◽  
Francois Richard
Keyword(s):  
Connexin 43 ◽  
In Vitro Maturation ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Gap Junctional

scholarly journals Lysophosphatidic acid inhibits gap-junctional communication and stimulates phosphorylation of connexin-43 in WB cells: possible involvement of the mitogen-activated protein kinase cascade

1994 ◽  
Vol 303 (2) ◽  
pp. 475-479 ◽  
Author(s):  
C S T Hill ◽  
S Y Oh ◽  
S A Schmidt ◽  
K J Clark ◽  
A W Murray
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Phorbol Ester ◽  
Connexin 43 ◽  
Mitogen Activated Protein Kinase ◽  
Western Blots ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Mitogen Activated Protein ◽  
Gap Junctional

Lysophosphatidic acid (LPA) was shown to be a powerful inhibitor of gap-junctional communication between cultured rat liver WB cells, as determined by the transfer of Lucifer Yellow, with 50% inhibition obtained at about 0.3 microM LPA. Inhibition of communication was rapid (5 min) and was maintained for at least 80 min. After incubation for 3 h with LPA, communication competence was partially restored and dye transfer was refractory to further addition of LPA. Communication in LPA-refractory cells retained sensitivity to inhibition by phorbol ester and by epidermal growth factor (EGF). LPA-induced inhibition was associated with phosphorylation of connexin-43 protein, as detected by slower migration of the protein detected on Western blots, which could be eliminated by incubation of samples with alkaline phosphatase. A close correspondence was observed between the time- and dose-dependency of LPA effects on communication and the induction of mitogen-activated protein kinase (MAP kinase). Activation of both the 42 kDa and 44 kDa subspecies were confirmed by mobility shifts on Western blots using an anti-(MAP kinase R1) (erk 1-III) antibody and by fractionation on Mono Q columns. Cells pretreated with phorbol ester for 24 h were insensitive to phorbol ester inhibition of communication or activation of MAP kinase, but retained their sensitivity to LPA. The results indicate that LPA initiates the activation of protein kinase cascades in WB cells that are probably independent of protein kinase C and identifies connexin-43 as one substrate for the activated kinases.

76 Effects of serum type in maturation medium on in vitro development of bovine embryos

2019 ◽  
Vol 31 (1) ◽  
pp. 163
Author(s):  
A. Mesalam ◽  
S. Zhang ◽  
K.-L. Lee ◽  
S.-H. Song ◽  
L. Xu ◽  
...  
Keyword(s):  
Lipid Content ◽  
Cumulus Cell ◽  
Cell Number ◽  
Mitochondrial Activity ◽  
Bovine Embryos ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Maturation Medium ◽  
Gap Junctional

This study investigated the effect of bovine serum albumin (BSA), charcoal:dextran stripped fetal bovine serum (CDS FBS), and heat-inactivated FBS (HI FBS) in maturation medium on their ability to support in vitro oocyte maturation, cumulus cell-oocyte gap junctional communication, and development of bovine embryos. Charcoal:dextran treatment of FBS removes lipophilic chemicals, certain steroid hormones, and certain growth factors; however, HI FBS have a lot-to-lot variation in steroid hormones level that can affect the reproducibility of experimental findings. Oocytes were cultured in TCM-199 supplemented with either 8% (w/v) BSA, 10% (v/v) CDS FBS, or 10% (v/v) HI FBS and 1µg mL−1 oestradiol-17β, 10µg mL−1 FSH, 10ng mL−1 epidermal growth factor, 0.6mM cysteine, 0.2mM sodium pyruvate, and followed by IVF, and the zygotes were cultured in SOF-BE1 medium. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, immunocytochemistry, and cryo-tolerance. The differences in embryo development between experimental groups were analysed by 1-way ANOVA. The Duncan’s multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P<0.05. We have shown that CDS FBS significantly improved (P<0.05) the percentage of MII oocytes compared with that in the BSA supplemented group (77.2±1.0% v. 69.3%±2.3%, respectively). Moreover, CDS FBS had a higher significant (P<0.05) effect on the rate of blastocyst formation compared with HI FBS and BSA (45.2±0.7% v. 37.4±1.5% and 31.1±3.9%, respectively; 6 replicates were performed). Culture of oocytes with CDS FBS increased (P<0.05) the expression of gap junction proteins, CX37 and CX43, at both transcriptional and translation levels as determined by quantitative RT-PCR and immunofluorescence analysis, respectively. We also found that CDS FBS significantly increased total cell number and decreased the apoptotic index in Day-8 blastocysts compared with the BSA group. Furthermore, the beneficial effects of CDS FBS on embryos were associated with significantly reduced intracellular lipid content and increased mitochondrial activity in both oocytes and blastocysts as identified by Nile red and MitoTracker Green staining, respectively. Taken together, these data suggest that supplementation of maturation medium with CDS FBS, as an alternative to HI FBS, affected cumulus cell-oocyte gap junctional communication, and subsequently improved in vitro developmental competence of bovine oocytes and embryos. Research was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant numbers: 117029-3 and 315017-5).

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