Cyclic AMP and LDL trigger a rapid enhancement in gap junction assembly through a stimulation of connexin trafficking

Given the rapid turnover of connexin proteins, gap junction (GJ) assembly represents an important means of regulating the extent of GJ communication between cells. This report describes an increase in the level of GJ assembly within one hour following treatment with cAMP-elevating reagents or low density lipoprotein (LDL). Dye transfer methods and freeze-fracture with electron microscopy were used to assay junctional permeability and structure, respectively, subsequent to the dissociation, recovery and reaggregation of Novikoff hepatoma cells. Reaggregating cells in the presence of agents that increase cAMP levels (8-Br-cAMP, forskolin and IBMX) enhanced both dye transfer rates between cells and the extent of GJ formation 2- to 3-fold. These data and studies with the protein kinase A inhibitor, H-89, indicate that cAMP signaling plays a key role in enhanced assembly. The response to LDL parallels that to cAMP and relies on the activity of both adenylyl cyclase and protein kinase A. Immunoblot analysis revealed no change in the level of connexin43 (Cx43) or its phosphorylation states over a period of 2.5 hours. However, three agents (brefeldin A, monensin and nocodazole), that inhibit intracellular membrane trafficking by different mechanisms, all blocked the enhanced assembly of GJs when triggered by either elevated cAMP or exposure to LDL. Related studies, which employed trafficking inhibitors at different stages in GJ assembly, suggested that Cx43 trafficking during enhanced assembly is regulated, in part, by cell contact. Intracellular sources of Cx43 were characterized by colabeling for several markers of cytoplasmic membrane systems. We conclude that an increase in GJ assembly: (i) occurs rapidly in the presence of elevated cAMP or LDL, (ii) does not require an increase in Cx43 levels or major changes in Cx43 phosphorylation and (iii) is dependent upon the trafficking of Cx43 from intracellular storage sites.

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Gap junction assembly: PTX-sensitive G proteins regulate the distribution of connexin43 within cells

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.4.c1211 ◽

2001 ◽

Vol 281 (4) ◽

pp. C1211-C1222 ◽

Cited By ~ 14

Author(s):

Paul D. Lampe ◽

Qiu Qiu ◽

Rita A. Meyer ◽

Erica M. TenBroek ◽

Timothy F. Walseth ◽

...

Keyword(s):

Plasma Membrane ◽

Gap Junction ◽

G Proteins ◽

Membrane Trafficking ◽

Low Density Lipoprotein ◽

Specific Inhibitor ◽

Density Lipoprotein ◽

Brefeldin A ◽

Dye Transfer ◽

Novikoff Hepatoma

Cells expressing connexin43 are able to upregulate gap junction (GJ) communication by enhancing the assembly of new GJs, apparently through increased connexin trafficking. Because G proteins are known to regulate different aspects of protein trafficking, we examined the effects of pertussis toxin (PTX; a specific inhibitor of certain G proteins) on GJ assembly. Dissociated Novikoff hepatoma cells were reaggregated for 60 min to form nascent junctions. PTX inhibited GJ assembly, as indicated by a reduction in dye transfer. Electron microscopy also revealed a 60% decrease in the number of GJ channels per cell interface. Importantly, PTX blocked the twofold enhancement in GJ assembly found in the presence of low-density lipoprotein. Two Giαproteins (Giα2 and Giα3), which have been implicated in the control of membrane trafficking, reacted with PTX in ADP-ribosylation studies. PTX and/or the trafficking inhibitors, brefeldin A and monensin, inhibited GJ assembly to comparable degrees. In addition, assays for GJ hemichannels demonstrated reduced plasma membrane levels of connexin43 following PTX treatment. These results suggest that PTX-sensitive G proteins regulate connexin43 trafficking, and, as a result of inhibition with PTX, the number of plasma membrane hemichannels available for GJ assembly is reduced.

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Protein kinase A-regulated membrane trafficking of a green fluorescent protein-aquaporin 5 chimera in MDCK cells

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/j.bbamcr.2006.02.005 ◽

2006 ◽

Vol 1763 (4) ◽

pp. 337-344 ◽

Cited By ~ 28

Author(s):

Chisato Kosugi-Tanaka ◽

Xuefei Li ◽

Chenjuan Yao ◽

Tetsuya Akamatsu ◽

Norio Kanamori ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Protein Kinase ◽

Protein Kinase A ◽

Membrane Trafficking ◽

Fluorescent Protein ◽

Mdck Cells ◽

Aquaporin 5 ◽

Green Fluorescent ◽

Kinase A

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Phosphorylation decreases trypsin activation and apolipoprotein AI binding to glycosylphosphatidylinositol-specific phospholipase D

Biochemistry and Cell Biology ◽

10.1139/o02-004 ◽

2002 ◽

Vol 80 (2) ◽

pp. 253-260 ◽

Cited By ~ 4

Author(s):

Mark A Deeg ◽

Rosario F Bowen

Keyword(s):

Protein Kinase ◽

Human Serum ◽

Protein Kinase A ◽

Phospholipase D ◽

Density Lipoprotein ◽

Fold Increase ◽

Mrna Levels ◽

Apolipoprotein Ai ◽

Kinase A

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is present in plasma as an apolipoprotein and as a cell-associated lipase. GPI-PLD mRNA levels are regulated, but it is unclear if posttranslational mechanisms also regulate GPI-PLD function. We examined the effect of protein kinase A phosphorylation on human serum GPI-PLD activity, trypsin activation, and apolipoprotein AI binding. Protein kinase A phosphorylation did not activate GPI-PLD activity in vitro, nor did phosphorylated GPI-PLD cleave a GPI-anchored protein from intact porcine erythrocytes. Trypsin cleaves the C-terminal β propeller of purified human serum GPI-PLD to generate three immunodetectable fragments (75, 28, and 18 kDa) in association with a 12-fold increase in enzyme activity. After phosphorylation, the amounts of 28- and 18-kDa fragments were markedly decreased with trypsin treatment, and activity was only increased five-fold. Phosphorylation also inhibits binding of GPI-PLD to apolipoprotein AI. These data are the first demonstrating that phosphorylation may regulate GPI-PLD interaction with other proteins.Key words: apolipoprotein AI, high-density lipoprotein, glycosylphosphatidylinositol, trypsin, phospholipase D.

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AKAP350 Interaction with cdc42 Interacting Protein 4 at the Golgi Apparatus

Molecular Biology of the Cell ◽

10.1091/mbc.e03-10-0757 ◽

2004 ◽

Vol 15 (6) ◽

pp. 2771-2781 ◽

Cited By ~ 47

Author(s):

M. Cecilia Larocca ◽

Ryan A. Shanks ◽

Lan Tian ◽

David L. Nelson ◽

Donn M. Stewart ◽

...

Keyword(s):

Protein Kinase ◽

Golgi Apparatus ◽

Protein Kinase A ◽

Membrane Trafficking ◽

Interacting Protein ◽

Anchoring Protein ◽

Regulatory Effects ◽

Related Proteins ◽

Kinase A

The A kinase anchoring protein 350 (AKAP350) is a multiply spliced type II protein kinase A anchoring protein that localizes to the centrosomes in most cells and to the Golgi apparatus in epithelial cells. In the present study, we sought to identify AKAP350 interacting proteins that could yield insights into AKAP350 function at the Golgi apparatus. Using yeast two-hybrid and pull-down assays, we found that AKAP350 interacts with a family of structurally related proteins, including FBP17, FBP17b, and cdc42 interacting protein 4 (CIP4). CIP4 interacts with the GTP-bound form of cdc42, with the Wiscott Aldrich Syndrome group of proteins, and with microtubules, and exerts regulatory effects on cytoskeleton and membrane trafficking. CIP4 is phosphorylated by protein kinase A in vitro, and elevation of intracellular cyclic AMP with forskolin stimulates in situ phosphorylation of CIP4. Our results indicate that CIP4 interacts with AKAP350 at the Golgi apparatus and that either disruption of this interaction by expressing the CIP4 binding domain in AKAP350, or reduction of AKAP350 expression by RNA interference leads to changes in Golgi structure. The results suggest that AKAP350 and CIP4 influence the maintenance of normal Golgi apparatus structure.

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Increased gap junction assembly between cultured cells upon cholesterol supplementation

Journal of Cell Science ◽

10.1242/jcs.96.2.231 ◽

1990 ◽

Vol 96 (2) ◽

pp. 231-238

Author(s):

R. Meyer ◽

B. Malewicz ◽

W.J. Baumann ◽

R.G. Johnson

Keyword(s):

Gap Junction ◽

Actinomycin D ◽

Cultured Cells ◽

Lucifer Yellow ◽

Freeze Fracture ◽

Cholesterol Content ◽

Dye Transfer ◽

Novikoff Hepatoma ◽

Fourfold Increase ◽

Cholesterol Supplementation

Novikoff hepatoma cells provide an excellent model system for the study of gap junction assembly, a process that could be influenced by lipids and other factors at numerous points. Since it is possible to alter the cellular levels of cholesterol in these cells, it was added to the cells in serum-supplemented medium and changes in gap junction assembly were evaluated. Cells were dissociated and reaggregated following exposure to a range of cholesterol concentrations for 24 h. A five- to sixfold increase in the number of aggregated gap junction particles and a 50% increase in cellular cholesterol content were observed with 20 microM added cholesterol. A 1-h exposure to added cholesterol, during cell reaggregation, resulted in a fourfold increase in the number of aggregated gap junction particles, demonstrating that the effect was rapid. The number of aggregated gap junction particles and formation plaque areas were used as measures of junction assembly and assayed by quantitative freeze-fracture and electron microscopy. Junctional permeabilities were evaluated by means of dye transfer times following the intracellular microinjection of Lucifer Yellow. Increased dye transfer was observed between cholesterol-treated cells, which suggested that the increase in assembly was accompanied by an increase in junction permeability. Cells were treated with cycloheximide (100 micrograms ml-1) and actinomycin D (10 micrograms ml-1) to determine whether protein and RNA syntheses were involved in the enhanced gap junction assembly. Cycloheximide but not actinomycin D blocked the increased junction assembly observed with added cholesterol. These results suggested that protein synthesis, but not RNA synthesis, is necessary for the increased gap junction formation observed.(ABSTRACT TRUNCATED AT 250 WORDS)

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Anchoring of Protein Kinase A-Regulatory Subunit IIα to Subapically Positioned Centrosomes Mediates Apical Bile Canalicular Lumen Development in Response to Oncostatin M but Not cAMP

Molecular Biology of the Cell ◽

10.1091/mbc.e06-08-0732 ◽

2007 ◽

Vol 18 (7) ◽

pp. 2745-2754 ◽

Cited By ~ 8

Author(s):

Kacper A. Wojtal ◽

Dick Hoekstra ◽

Sven C.D. van IJzendoorn

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Membrane Trafficking ◽

Oncostatin M ◽

Regulatory Subunit ◽

Apical Surface ◽

Dependent Manner ◽

Anchoring Protein ◽

Pka Regulatory Subunit ◽

Kinase A

Oncostatin M and cAMP signaling stimulate apical surface-directed membrane trafficking and apical lumen development in hepatocytes, both in a protein kinase A (PKA)-dependent manner. Here, we show that oncostatin M, but not cAMP, promotes the A-kinase anchoring protein (AKAP)-dependent anchoring of the PKA regulatory subunit (R)IIα to subapical centrosomes and that this requires extracellular signal-regulated kinase 2 activation. Stable expression of the RII-displacing peptide AKAP-IS, but not a scrambled peptide, inhibits the association of RIIα with centrosomal AKAPs and results in the repositioning of the centrosome from a subapical to a perinuclear location. Concomitantly, common endosomes, but not apical recycling endosomes, are repositioned from a subapical to a perinuclear location, without significant effects on constitutive or oncostatin M-stimulated basolateral-to-apical transcytosis. Importantly, however, the expression of the AKAP-IS peptide completely blocks oncostatin M-, but not cAMP-stimulated apical lumen development. Together, the data suggest that centrosomal anchoring of RIIα and the interrelated subapical positioning of these centrosomes is required for oncostatin M-, but not cAMP-mediated, bile canalicular lumen development in a manner that is uncoupled from oncostatin M-stimulated apical lumen-directed membrane trafficking. The results also imply that multiple PKA-mediated signaling pathways control apical lumen development and that subapical centrosome positioning is important in some of these pathways.

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