Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cardiac gap junctions permit the conduction and propagation of the electrical impulses that are responsible for the synchronous contraction of the myocardium (Page and Manjunath, 1986). Cardiac gap junctions are formed by the association of connexin molecules within the plasma membrane of heart cells. They create aqueous channels that permit exchange of ions and other small molecules between adjacent cells. Intercellular communication via these channels may be regulated by phosphorylation. cAMP was shown to increase junctional conductance and stimulate phosphorylation of connexin32 in cultures of dissociated liver hepatocytes (Saez et al., 1986). Furthermore, a 47 kDa protein purified from dog heart gap junctions was phosphorylated in vitro by the catalytic subunit of protein kinase A (Pressler and Hathaway, 1987). Other studies have demonstrated that cAMP enhanced junctional conductance in intact heart and isolated heart cells (De Mello, 1986; De Mello and van Loon, 1987; Burt and Spray, 1988). This report provides direct evidence that the heart gap junction protein, connexin43, from unstimulated heart tissues and cultured myocytes is phosphorylated stoichiometrically in vivo. Phosphorylation of 45 and 47 kDa connexin43-related proteins occurred predominantly on serine. In addition, the 47 kDa protein contained a low-level of phosphothreonine.
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PMID:Evidence that heart connexin43 is a phosphoprotein. 165 41

The effects of cAMP-dependent protein kinase A and protein kinase C on cell-cell communication have been examined in primary ovarian granulosa cells microinjected with purified components of these two regulatory cascades. These cells possess connexin43 (alpha 1)-type gap junctions, and are well-coupled electrotonically and as judged by the cell-to-cell transfer of fluorescent dye. Within 2-3 min after injection of the protein kinase A inhibitor (PKI) communication was sharply reduced or ceased, but resumed in about 3 min with the injection of the protein kinase A catalytic subunit. A similar resumption also occurred in PKI-injected cells after exposure to follicle stimulating hormone. Microinjection of the protein kinase C inhibitor protein caused a transient cessation of communication that spontaneously returned within 15-20 min. Treatment of cells with activators of protein kinase C, TPA or OAG for 60 min caused a significant reduction in communication that could be restored within 2-5 min by the subsequent injection of either the protein kinase C inhibitor or the protein kinase A catalytic subunit. With a longer exposure to either protein kinase C activator communication could not be restored and this appeared to be related to the absence of aggregates of connexin43 in membrane as detected immunologically. In cells injected with alkaline phosphatase communication stopped but returned either spontaneously within 20 min or within 2-3 min of injecting the cell with either the protein kinase A catalytic subunit or with protein kinase C. When untreated cells were injected with protein kinase C communication diminished or ceased within 5 min. Collectively these results demonstrate that cell-cell communication is regulated by both protein kinase A and C, but in a complex interrelated manner, quite likely by multiple phosphorylation of proteins within or regulating connexin-43 containing gap junctions.
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PMID:In situ regulation of cell-cell communication by the cAMP-dependent protein kinase and protein kinase C. 793 58

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.
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PMID:Lysophosphatidic acid inhibits gap-junctional communication and stimulates phosphorylation of connexin-43 in WB cells: possible involvement of the mitogen-activated protein kinase cascade. 798 Apr 7

One important role of the junctional communication in the ovarian follicle is to mediate transmission of cAMP, the regulatory signal that maintains the oocyte in meiotic arrest. Luteinizing hormone (LH) interrupts cell-to-cell communication within the ovarian follicle, leading to a decrease in intraoocyte concentrations of cAMP followed by resumption of meiosis. Our experiments were directed at exploration of mechanisms involved in the LH-induced communication breakdown in the preovulatory ovarian follicle. Immunofluorescence and Western blot analysis, using highly specific antibodies, showed that connexin-43 (Cx43), the ovarian gap junction protein, is present in the cytoplasmic membranes of the follicular cells in multiple phosphorylated forms. The relative amounts of the different forms of Cx43 vary in response to LH: short time exposure (10 min) stimulated phosphorylation of Cx43 followed by immediate dephosphorylation, while longer incubations (8 and 24 h) with this hormone resulted in elimination of the protein. Forskolin mimicked the LH-induced phosphorylation/dephosphorylation, as well as the decrease of Cx43 protein level. A gonadotropin-releasing hormone analog (GnRHa) also induced an immediate phosphorylation/dephosphorylation of Cx43 and a later reduction of the amount of Cx43. The direct PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced phosphorylation of Cx43 that was completely blocked by the protein kinase C inhibitor, staurosporine. This kinase inhibitor partially interfered with LH, but not forskolin-induced phosphorylation of Cx43. Analysis of the effect of LH on Cx43 gene expression revealed a significant decrease (45%) in Cx43 mRNA level at 24 h of incubation. A drop of Cx43 mRNA was also induced by GnRHa. Our results suggest that the LH-induced gating mechanism of the gap junctions in rat ovarian follicles is comprised of two steps: the immediate response is represented by a change in the phosphorylation state of the Cx43 protein, and the later response is manifested by a reduction of Cx43 protein level, due to attenuation of its gene expression. Phosphorylation of Cx43 may occur through PKA-, as well as PKC-dependent pathways.
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PMID:Phosphorylation and expression of connexin-43 ovarian gap junction protein are regulated by luteinizing hormone. 798 67

Human myometrium contains hCG/LH receptors. There are fewer of these receptors during labor compared to no labor at preterm or term deliveries. Exogenous hCG can directly inhibit oxytocin-stimulated human myometrial contractions. These findings suggest that hCG may directly maintain myometrial quiescence during pregnancy. As maintenance of uterine quiescence may involve down-regulation of myometrial gap junctions, we investigated the effect of hCG on connexin-43 (CX-43) gene expression from RNA to protein and morphological gap junctions. The addition of 5 or 10 nM highly purified hCG to subconfluent cultures of pregnant myometrial smooth muscle cells resulted in a significant decrease in CX-43 protein levels. Higher hCG concentrations (100 and 1000 nM), however, had no effect. The maximal effect of hCG was seen at 4-8 h of culture, followed by recovery after a longer duration of culture. hCG treatment also concomitantly decreased CX-43 messenger RNA and morphological gap junctions. The hCG effect on CX-43 protein levels is hormone specific and mediated by protein kinase-A signaling. Estradiol and oxytocin increased, whereas progesterone decreased, CX-43 protein levels and morphological gap junctions. The oxytocin-induced increase was reversed by cotreatment with hCG. Although RU 486 alone had no effect on CX-43 protein levels, it prevented the down-regulating action of hCG and progesterone. In summary, our results demonstrate that hCG can directly decrease CX-43 messenger RNA, protein, and morphological gap junctions in cultured pregnant human myometrial smooth muscle cells. The hCG action is concentration and time dependent, hormone specific, and mediated by protein kinase-A signaling and appears to involve progesterone receptors. These data lend support to the concept that hCG could be one of the hormones responsible for maintaining uterine quiescence by down-regulating myometrial gap junctions during pregnancy.
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PMID:Novel regulation of pregnant human myometrial smooth muscle cell gap junctions by human chorionic gonadotropin. 798 70

Isolated connexin-32s from rat and mouse liver are proteolyzed in vitro by the intracellular Ca(2+)-dependent neutral proteases, mu-calpain and m-calpain, producing a major fragment of 26 kDa. Connexin-26 is not proteolyzed by calpain. Calpain cleaves connexin-32 at its C-terminal end as shown by 125I-calmodulin binding experiments. Connexin-32, but not connexin-26, is phosphorylated by both protein kinase A and protein kinase C in serine residues and the sites of phosphorylation by both kinases remain in the major 26-kDa fragment resulting from calpain proteolysis. Phosphorylation of connexin-32 by protein kinase C, but not by protein kinase A, prevents the proteolytic attack of mu-calpain and m-calpain. Phosphorylation of connexin-32 by protein kinase A and protein kinase C does not prevent its proteolysis by papain, alpha-chymotrypsin, proteinase K, and trypsin.
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PMID:Phosphorylation of connexin-32 by protein kinase C prevents its proteolysis by mu-calpain and m-calpain. 839 Sep 88

Purification of the lens fiber cell membrane proteins MP20 and MP26, and the partial co-purification of the lens connexin-related proteins MP70 and connexin 46 has been achieved using anion- and cation-exchange chromatography of lens fiber cell membrane proteins solubilized in n-octyl-beta-D-glucopyranoside (octyl glucoside). The apparent molecular weights of the solubilized protein-detergent complexes were significantly greater than that expected for the monomeric proteins. The purified proteins retained their ability to be phosphorylated by cAMP-dependent protein kinase, and to bind calmodulin in a calcium and magnesium dependent manner. The heterobifunctional covalent chemical crosslinking agent N-5-azido-2-nitro-benzoyloxysuccinimide (ANB-NOS), and the thiol oxidant cupric phenanthroline were used to identify the oligomeric states of these proteins. Crosslinking of either the purified proteins or native lens membranes generated a ladder of crosslinked MP20 or MP26 homo-oligomers. The largest detectable crosslinked homo-oligomer of MP20 was at least a hexamer, while for MP26 the largest crosslinked homo-oligomer was at least a tetramer. The possible oligomeric states of MP70 and connexin 46 could not be determined with the crosslinking reagents used in this study. The procedure described here for the purification of detergent-solubilized major lens proteins should provide a valuable approach in future studies aimed at clarifying the roles of these different lens membrane proteins.
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PMID:Purification and oligomeric state of the major lens fiber cell membrane proteins. 852 19

The cAMP-dependent protein-kinase-catalyzed phosphorylation of the two major intrinsic lens fiber cell plasma membrane proteins, MP20 and MP26, is likely restricted to the inner cortical and nuclear regions of the lens in vivo. The ovine-lens-specific connexin, MP70, that has been identified as Cx50 in mice and Cx45.6 in the chick, is also a protein kinase substrate although it does not appear to be phosphorylated by a number of protein kinases including cAMP-dependent protein kinase, calmodulin-dependent protein kinase or protein kinase C. Rather, an extrinsic lens membrane fraction was isolated which contained protein kinase activity that catalyzed the phosphorylation of MP70; this protein kinase activity was cAMP-independent, Ca(2+)-independent, Mg(2+)-dependent, phosphorylated MP70 on a serine residue(s) and migrated with a molecular mass of 35 kDa on a gel filtration column. Both MP70 phosphorylation and the endogenous protein kinase activity were restricted to the lens outer cortical region. This membrane-associated protein kinase activity represents the first reported partial characterization of an endogenous lens fiber cell protein kinase activity that catalyzes the phosphorylation of a lens connexin protein. The phosphatase-induced shift in the electrophoretic mobility of MP70 is not reversed by this protein kinase, indicating that MP70 is likely phosphorylated on different residues by two or more protein kinases.
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PMID:Characterization of the ovine-lens plasma-membrane protein-kinase substrates. 853 18

We have used low stringency hybridization to clone a novel connexin from a skate retinal cDNA library. A rat connexin 32 clone was used to isolate a single partial clone that was subsequently used to isolate seven more overlapping clones of the same cDNA. Two clones containing the entire open reading frame have a consensus sequence of 1456 bp and predict a protein of 302 amino acids length and molecular mass of 35,044 daltons, referred to as connexin 35 or Cx35. Southern blot analysis suggests that the cloned sequence lies in a single gene with one intron. Polymerase chain reaction amplification from genomic DNA and partial sequencing of this intron showed that it was approximately 950 bp in length, and located within the coding region 71 bp after the translation start site. Hydropathy analysis of the predicted protein and alignments with previously cloned connexins indicate that Cx35 has a long cytoplasmic loop and a relatively short carboxyl terminal tail. Multiple sequence alignments show that Cx35 has similarities to both alpha and beta groups of connexins and suggests that its origins may be near the divergence point for the two groups. Consensus sequences consistent with sites for phosphorylation by protein kinase C and by cAMP - or cGMP -dependent protein kinase were identified. Two transcripts were detected in Northern blot analysis: a 1.95-kb primary transcript and a 4.6-kb minor transcript. In RNA samples from 10 tissues, transcripts were detected only in the retina.
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PMID:Connexin 35: a gap-junctional protein expressed preferentially in the skate retina. 868 55

The lens gap-junction protein, connexin 56, is modified by phosphorylation. Two-dimensional mapping of tryptic phosphopeptides of 32P-labeled connexin 56 from primary chicken-lens cultures showed that treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) induced an increase in phosphorylation of connexin 56 at specific constitutively phosphorylated sites. Treatment with 8-Br-cAMP or forskolin did not induce substantial changes in connexin 56 phosphorylation. Two phosphorylation sites within connexin 56, S493 and S118, were identified after HPLC purification and peptide sequencing of tryptic phosphopeptides from bacterially expressed connexin 56 fusion proteins phosphorylated by protein kinase C or protein kinase A in vitro. Comparisons of the two-dimensional maps of tryptic phosphopeptides from in vitro phosphorylated connexin 56 fusion proteins and in vivo phosphorylated connexin 56 showed that S493 and S118 were constitutively phosphorylated in lentoid-containing cultures, and that treatment with TPA induced an increase in phosphorylation of the peptides containing S118. It is suggested that phosphorylation of connexin 56 at S118 is involved in the TPA-induced decrease in intercellular communication and acceleration of connexin 56 degradation.
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PMID:The gap-junction protein connexin 56 is phosphorylated in the intracellular loop and the carboxy-terminal region. 906 50


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