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)

The appropriate functioning of tissues and organ systems depends on intercellular communication such as gap junctions formed by connexin (Cx) protein channels between adjacent cells. We have previously shown that Swiss 3T3 cells aggregated on hydrophilic cellulose substratum Cuprophan (CU) establish short linear gap junctions composed of Cx 43 in cell surface plaques. This phenomenon seems to depend on the high intracellular cyclic AMP (cAMP) concentration triggered by attachment of the cells to CU. We have now used a cellulose-coated polystyrene inducing the same cell behaviour to analyse the gap junction communication between aggregated cells. The transfer of the dye Lucifer Yellow (LY) between cells showed that cells aggregated on cellulose substratum rapidly (within 90 min) establish functional gap junctions. Inhibitors of cAMP protein kinase (PKI) or protein kinase C (GF109203X) both inhibited the diffusion of LY between neighbouring cells. Western blot analysis showed that this change in permeability was correlated with a decrease in Cx 43 phosphorylation. Thus, cellulose substrata seem to induce cell-cell communication through Cx 43 phosphorylation modulated by PKA and PKC. To understand the mechanisms by which a substratum regulates gap junctional communication is critically important for the emerging fields of tissue engineering and biohybrid devices.
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PMID:Gap junction communication between cells aggregated on a cellulose-coated polystyrene: influence of connexin 43 phosphorylation. 1475 34

Gap junctions are specialized membrane domains composed of collections of channels that directly connect neighboring cells providing for the cell-to-cell diffusion of small molecules, including ions, amino acids, nucleotides, and second messengers. Vertebrate gap junctions are composed of proteins encoded by the "connexin" gene family. In most cases examined, connexins are modified post-translationally by phosphorylation. Phosphorylation has been implicated in the regulation of gap junctional communication at several stages of the connexin "lifecycle", such as the trafficking, assembly/disassembly, degradation, as well as, the gating of gap junction channels. Since connexin43 (Cx43) is widely expressed in tissues and cell lines, we understand the most about how it is regulated, and thus, connexin43 phosphorylation is a major focus of this review. Recent reports utilizing new methodologies combined with the latest genome information have shown that activation of several kinases including protein kinase A, protein kinase C, p34(cdc2)/cyclin B kinase, casein kinase 1, mitogen-activated protein (MAP) kinase and pp60(src) kinase can lead to phosphorylation at 12 of the 21 serine and two of the six tyrosine residues in the C-terminal region of connexin43. In several cases, use of site-directed mutants of these sites have shown that these specific phosphorylation events can be linked to changes in gap junctional communication.
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PMID:The effects of connexin phosphorylation on gap junctional communication. 1510 65

Gap junction channels form the basis of intercellular communication in the heart. In the working myocardium, the connexin43 (Cx43) is most abundantly found, whereas connexin40 (Cx40) is expressed in the atria and in the conduction system [together with low levels of connexin45 (Cx45)]. However, little is known about the differential regulation of the connexins by pathophysiologically stimuli such as tumor necrosis factor alpha (TNFalpha). Inasmuch as TNFalpha may play a contributory role in the concert of factors involved in the pathophysiology of heart failure and because this cardiac disease often leads to ventricular reentrant arrhythmia, the goal of our study was to find out whether TNFalpha may influence the expression of the cardiac connexins connexin43, connexin40, and connexin45. Neonatal rat cardiomyocytes were exposed to TNFalpha (10, 40, 100, 400, and 1000 pg/ml) for 24 h with or without additional treatment with the mitogenic-activated protein kinase (MAP-kinase) inhibitors SB203580 [4-(4-fluorophenyl)-2-(4-methyl-sulfinylphenyl)-5-(4-pyridyl)-1H-imidazole; 10(-5) M, protein38 mitogenic-activated protein kinase (p38 MAP kinase) inhibitor] or the MEK1 (mitogenic-activated protein kinase/extracellular signal-regulated kinase kinase) inhibitor PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; 10(-5) M]. Connexin43, connexin40, and connexin45 expressions were analysed using Western blot analysis, immunohistology, and polymerase chain reaction (PCR) studies (connexin43 and connexin40). TNFalpha induced a concentration-dependent increase in connexin43 (by 2.9+/-0.6, P<0.05, n=5) but not in connexin40 or connexin45 expressions. Both connexins (40 and 45) showed a very low expression near the detection limit. The increases in connexin43 expression could be completely suppressed by SB203580 (0.9+/-0.4, P<0.05, n=5) but not by PD98059. In absence of a stimulating drug, these inhibitors (SB203580 or PD98059) did not affect connexin43 content. Additional PCR experiments revealed increases in connexin43 mRNA under the influence of 100 pg/ml TNFalpha (211+/-38%, P<0.05, n=5), which could be completely suppressed by SB203580. In contrast, the connexin40 expression remained unchanged. From these results, we conclude that TNFalpha can differentially regulate cardiac connexin expression via p38 MAP kinase pathway and thus may alter intercellular communication. This may contribute to the changes observed in heart failure with regard to the formation of an arrhythmogenic substrate.
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PMID:Chronic regulation of the expression of gap junction proteins connexin40, connexin43, and connexin45 in neonatal rat cardiomyocytes. 1549 89

Osteoblasts are highly coupled by gap junctions formed by connexin43. Overexpression of connexin45 in osteoblasts results in decreased chemical and electrical coupling and reduces gene transcription from connexin response elements (CxREs) in the osteocalcin and collagen Ialpha1 promoters. Here, we demonstrate that transcription from the gap junction-dependent osteocalcin CxRE is regulated by extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol 3-kinase (PI3K) cascades. Overexpression of a constitutively active mitogen-activated protein kinase kinase (MEK), Raf, or Ras can increase transcription more than twofold of the CxRE, whereas inhibition of MEK or PI3K can decrease transcription threefold from the osteocalcin CxRE. Importantly, disruption of gap junctional communication by overexpression of connexin45 or treatment with pharmacological inhibitors of gap junctions results in reduced Raf, ERK, and Akt activation. The consequence of attenuated gap junction-dependent signal cascade activation is a decrease in Sp1 phosphorylation by ERK, resulting in decreased Sp1 recruitment to the CxRE and inhibited gene transcription. These data establish that ERK/PI3K signaling is required for the optimal elaboration of transcription from the osteocalcin CxRE, and that disruption of gap junctional communication attenuates the ability of cells to respond to an extracellular cue, presumably by limiting the propagation of second messengers among adjacent cells by connexin43-gap junctions.
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PMID:Gap junctions regulate extracellular signal-regulated kinase signaling to affect gene transcription. 1552 79

Gap junctions, composed of proteins from the connexin family, allow for intercellular communication between cells and are important in development and maintenance of cell homeostasis. Phosphorylation has been implicated in the regulation of gap junctional communication at several stages of the cell cycle and the connexin "lifecycle", such as trafficking, assembly/disassembly, degradation, as well as in the gating of "hemi" channels or intact gap junction channels. This review focuses on how phosphorylation can regulate the early stages of the connexin life cycle through assembly of functional gap junctional channels. The availability of sequences from the human genome databases has indicated that the number of connexins in the gene family is approximately 20, but we know mostly about how connexin43 (Cx43) is regulated. Recent technologies and investigations of interacting proteins have shown that activation of several kinases including protein kinase A, protein kinase C (PKC), p34(cdc2)/cyclin B kinase, casein kinase 1 (CK1), mitogen-activated protein kinase (MAPK) and pp60(src) kinase can lead to phosphorylation of the majority of the 21 serine and two of the tyrosine residues in the C-terminal region of Cx43. While many studies have correlated changes in kinase activity with changes in gap junctional communication, further research is needed to directly link specific phosphorylation events with changes in connexin oligomerization and gap junction assembly.
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PMID:Connexin phosphorylation as a regulatory event linked to gap junction channel assembly. 1595

Although electrical coupling along the arteriolar endothelium is central in arteriolar conducted response and in control of vascular resistance, little is known about the pathophysiological effect of hypoxia and reoxygenation (H/R) on this coupling. We examined this effect in a monolayer of cultured microvascular endothelial cells (ECs) derived from wild-type (WT) or connexin (Cx)40-/- mice (Cx40 is a key gap junction protein in ECs). To assess electrical coupling, we used a current injection technique and Bessel function model to compute the monolayer intercellular resistance. Hypoxia (0.1% O2, 1 h) followed by abrupt reoxygenation (5-90 min) reduced coupling (i.e., increased resistance) in WT but not in Cx40-/- monolayer. H/R increased superoxide production and reduced protein kinase A (PKA) activity in both monolayers. Activation of PKA by 8-bromo-cAMP prevented the reduction in coupling. Preloading of the WT monolayer with the antioxidant ascorbate prevented reductions in both PKA activity and cell coupling. Inhibition of PKA with 6-22 amide during normoxia mimicked the reduction in coupling. Finally, hypoxia followed by slow reoxygenation caused no change in superoxide level, PKA activity, or coupling. Using intravital microscopy, we assessed the physiological relevance of these findings in terms of KCl-induced conducted vasoconstriction in arterioles of WT mouse cremaster muscle in vivo. Ischemia (1 h) followed by abrupt reperfusion (15-30 min) reduced conduction. 8-bromo-cAMP prevented this reduction, while 6-22 amide mimicked this reduction in control nonischemic arterioles. We propose that abrupt reoxygenation reduces interendothelial electrical coupling via oxidant- and PKA-dependent signaling that targets Cx40. We suggest that this mechanism contributes to compromised arteriolar function after H/R.
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PMID:Abrupt reoxygenation following hypoxia reduces electrical coupling between endothelial cells of wild-type but not connexin40 null mice in oxidant- and PKA-dependent manner. 1603 99

The umbrella cells that line the bladder are mechanosensitive, and bladder filling increases the apical surface area of these cells; however, the upstream signals that regulate this process are unknown. Increased pressure stimulated ATP release from the isolated uroepithelium of rabbit bladders, which was blocked by inhibitors of vesicular transport, connexin hemichannels, ABC protein family members, and nucleoside transporters. Pressure-induced increases in membrane capacitance (a measure of apical plasma membrane surface area where 1 microF approximately equals 1 cm2) were inhibited by the serosal, but not mucosal, addition of apyrase or the purinergic receptor antagonist PPADS. Upon addition of purinergic receptor agonists, increased capacitance was observed even in the absence of pressure. Moreover, knockout mice lacking expression of P2X2 and/or P2X3 receptors failed to show increases in apical surface area when exposed to hydrostatic pressure. Treatments that prevented release of Ca2+ from intracellular stores or activation of PKA blocked ATPgammaS-stimulated changes in capacitance. These results indicate that increased hydrostatic pressure stimulates release of ATP from the uroepithelium and that upon binding to P2X and possibly P2Y receptors on the umbrella cell, downstream Ca2+ and PKA second messenger cascades may act to stimulate membrane insertion at the apical pole of these cells.
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PMID:ATP and purinergic receptor-dependent membrane traffic in bladder umbrella cells. 1611 Mar 27

Gap junctions, composed of proteins from the connexin family, allow for intercellular communication between cells in tissues and are important in development, tissue/cellular homeostasis, and carcinogenesis. Genome databases indicate that there are at least 20 connexins in the mouse and human. Connexin phosphorylation has been implicated in connexin assembly into gap junctions, gap junction turnover, and cell signaling events that occur in response to tumor promoters and oncogenes. Connexin43 (Cx43), the most widely expressed and abundant gap junction protein, can be phosphorylated at several different serine and tyrosine residues. Here, we focus on the dynamic regulation of Cx43 phosphorylation in tissue and how these regulatory events are affected during development, wound healing, and carcinogenesis. The activation of several kinases, including protein kinase A, protein kinase C, p34cdc2/cyclin B kinase, casein kinase 1, mitogen-activated protein kinase, and pp60src kinase, can lead to the phosphorylation of different residues in the C-terminal region of Cx43. The use of antibodies specific for phosphorylation at defined residues has allowed the examination of specific phosphorylation events both in tissue culture and in vivo. These new antibody tools and those under development will allow us to correlate specific phosphorylation events with changes in connexin function.
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PMID:Temporal regulation of connexin phosphorylation in embryonic and adult tissues. 1613 42

Wild type connexin 46 of rat (wtrCx46), and human connexin 26 (wthCx26) and derivates from rCx46 elongated at the C-terminus by 25 amino acids (rCx46Ct) as well as C-terminal truncated constructs (rCx28.1, rCx45.3) were expressed in frog oocytes of Xenopus laevis. Single oocyte voltage-clamp analysis revealed that connexons or hemichannels of rCx46Ct exhibit similar conducting properties as those of wtrCx46. Insertion of a stop codon at C-terminal domains at position 243 and 409 resulted in a significant reduction in the corresponding hemichannel conductance. This result was also found for wthCx26, the shortest human connexin. Tagged connexin constructs rCx46Ct and hCx26Ct could be expressed in E. coli as monomers. The monomers of rCx46Ct and hCx26Ct were purified and electro-eluted from corresponding SDS gels. Studies of in vitro oligomerization showed that hexamers of these connexins were formed in presence of kinase and specific lipids. Purified rCx46Ct formed some oligomers in vitro if a lipid mixture of POPE/POPG and casein kinase I (CKI) was added, but in the presence of POPC, phosphorylated rCx46Ct monomers preferentially formed hexamers. Purified hCx26Ct formed hexamers in the presence of POPE/POPG. In addition, N-terminal truncated rCx46 (Cx35) oligomerized after phosphorylation. Reconstitution of purified recombinant connexin rCx46Ct in planar lipid bilayers mediated Ca(2+)-sensitive single channel activity. It is discussed whether the specific C-terminal end of the expressed connexins are responsible for hexamer formation as well as channel opening.
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PMID:Length of C-terminus of rCx46 influences oligomerization and hemichannel properties. 1638 79

We have been investigating the function and gene expression of connexins by vascular wall cells, especially Connexin43 (Cx43) in bovine aortic endothelial cells (BAEC). In this study, we tested the effects of carbenoxolone (CBN), a gap junction communication (GJC) blocker on the junctional transfer of Lucifer yellow in BAEC. CBN is a water-soluble derivative of the liquorice-root extract 18-alpha-glycyrrhetinic acid. CBN rapidly abolished dye-transfer in the scrape-load transfer assay (a measure of GJC) in a reversible and dose-dependent fashion. We then asked whether the BAEC might somehow compensate for the loss of junctional communication by altering the expression of connexins. Thus, we treated BAEC with 100 microM CBN in serum free medium and determined the total Cx43 cellular distribution (immunostaining) and protein content (immunoblotting). Besides changes in distribution, by 6 h, Cx43 content levels increased to 166% +/- 22% (P < 0.0001) of controls. RNA blot data showed two-three fold increases in Cx43 message in BAEC after 6 h of CBN treatment, suggesting transcriptional control. Since CBN has structural similarities to corticosteroids, we tested both aldosterone and prednisolone but neither drug increased Cx43 levels, suggesting that the CBN response was not due to a generalized steroid effect. Staurosporine inhibited the CBN-induced increase in Cx43 content, suggesting a role for kinases in the signaling pathway. Further studies with inhibitors indicated that PKA but not PKC was implicated. In summary, CBN blocks junctional communication and modulates Cx43 expression in BAEC. These results suggest a feedback mechanism for control of connexin expression based on junctional patency.
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PMID:Carbenoxolone inhibits junctional transfer and upregulates Connexin43 expression by a protein kinase A-dependent pathway. 1655 23


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