Identification of Isoforms of G Proteins and PKC that Colocalize with Tight Junctions

Dodane, V.; Kachar, Bechara

doi:10.1007/s002329900020

Cited by 119 publications

(63 citation statements)

References 35 publications

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“…1D). It was previously shown in separate studies that G␣ 12 and PP2A are localized in epithelial cell tight junctions (21,22). Endogenous G␣ 12 and A␣ were colocalized by immunofluorescent microscopy using antibodies to G␣ 12 and A␣ in Caco-2 cells and primary cultured neurons (Fig.…”

Section: Resultsmentioning

confidence: 67%

Gα12 Directly Interacts with PP2A

Zhu

Kosik

Meigs

et al. 2004

Journal of Biological Chemistry

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in ϳ300% stimulation of PP2A activity that was not detected with other G␣ subunits and was similar with GTP␥S-and GDP-liganded G␣ 12 . When tau and active kinase (Cdk5 and p25) were cotransfected in to COS cells, there was robust tau phosphorylation. Co-expression of wild type or QL␣ 12 with tau and the active kinase resulted in 60 ؎ 15% reductions in tau phosphorylation. In primary cortical neurons stimulated with lysophosphatitic acid, a 50% decrease in tau phosphorylation was observed. The G␣ 12 effect on tau phosphorylation was inhibited by the PP2A inhibitor, okadaic acid (50 nM), in COS cells and neurons. Taken together, these findings reveal novel, direct regulation of PP2A activity by G␣ 12 and potential in vivo modulation of PP2A target proteins including tau.Heterotrimeric G proteins 1 regulate many fundamental cellular responses, and the canonical receptor-G protein-effector paradigm has become significantly more complex in recent years. G protein signaling is modulated through interactions with families of regulatory proteins that affect nucleotide binding and hydrolysis. These include the RGS (regulators of G protein signaling) protein family that stimulate G␣ GTPase activity (reviewed in Ref. 1), and the GPR (G protein regulatory) protein family that share a conserved motif that inhibits GDP release from the G␣ i /␣ o families of G␣ subunits (2-4). G␣ 12/13 have multiple cellular functions including regulation of the actin cytoskeleton (5) and many functions are shared by both G␣ 12 and G␣ 13 subunits. G␣ 12/13 regulation of the actin cytoskeleton and stress fiber formation occurs through direct interactions with Rho regulatory proteins (6, 7), and other aspects of G␣ 12 /␣ 13 signaling are regulated through specific interactions with membrane or scaffolding proteins. For example, the binding of G␣ 12 and G␣ 13 to the C terminus of E-cadherin displaces ␤-catenin permitting it to signal (8, 9). We identified direct binding of both wild type and constitutively active (QL)␣ 12 to ZO-1 and regulation of paracellular permeability of Madin-Darby canine kidney cells (10,11). In addition, G␣ 12 interacts with AKAP-lbc, a scaffolding molecule that organizes components of cAMP signaling and Rho signaling machinery (12). Here, we have identified the A␣ subunit of Ser/ Thr phosphatase PP2A as another "scaffolding" protein that selectively interacts with G␣ 12 .Tau is a microtubule-associated protein that is predominantly expressed in neurons and functions to stabilize the cytoskeleton. Tau phosphorylation is necessary for microtubule binding and other protein interactions, but hyperphosphorylated tau is the predominant component of neurofibrillary tangles, a pathologic hallmark finding found in several neurodegenerative disorders including Alzheimer disease and FTDP-17 (frontotemporal dementia and Parkinson's disease linked to chromosome 17; reviewed in Ref. 13). Several kinases phosphorylate tau including mitogen-activated protein kinase (MAP), glycogen synthase 3␤ (GSK-3␤), tau-tubulin kinase, and cyclindepe...

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Section: Resultsmentioning

confidence: 67%

Gα12 Directly Interacts with PP2A

Zhu

Kosik

Meigs

et al. 2004

Journal of Biological Chemistry

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“…In these TJ domain regions, nPKC-y will be localized next to a TJ protein substrate (e.g., claudin, occludin, ZO-1) and thus in a position to modify TJ Ser/Thr phosphorylation status leading to altered BBB function. Supporting this hypothesis, in vitro studies have shown that aPKC-z colocalizes with ZO-1 in both Madin-Darby canine kidney and in human colon adenocarcinoma cell lines (Dodane and Kachar, 1996). Banan et al (2007) have shown that nPKC-y isozymes modify claudin isotypes resulting in altered intestinal epithelium barrier properties.…”

Section: Discussionmentioning

confidence: 89%

Protein Kinase C Activation Modulates Reversible Increase in Cortical Blood–Brain Barrier Permeability and Tight Junction Protein Expression during Hypoxia and Posthypoxic Reoxygenation

Willis

Meske

Davis

2010

J Cereb Blood Flow Metab

100

101

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Hypoxia (Hx) is a component of many disease states including stroke. Ischemic stroke occurs when there is a restriction of cerebral blood flow and oxygen to part of the brain. During the ischemic, and subsequent reperfusion phase of stroke, blood-brain barrier (BBB) integrity is lost with tight junction (TJ) protein disruption. However, the mechanisms of Hx and reoxygenation (HR)-induced loss of BBB integrity are not fully understood. We examined the role of protein kinase C (PKC) isozymes in modifying TJ protein expression in a rat model of global Hx. The Hx (6% O 2 ) induced increased hippocampal and cortical vascular permeability to 4 and 10 kDa dextran fluorescein isothiocyanate (FITC) and endogenous rat-IgG. Cortical microvessels revealed morphologic changes in nPKC-h distribution, increased nPKC-h and aPKC-f protein expression, and activation by phosphorylation of nPKC-h (Thr538) and aPKC-f (Thr410) residues after Hx treatment. Claudin-5, occludin, and ZO-1 showed disrupted organization at endothelial cell margins, whereas Western blot analysis showed increased TJ protein expression after Hx. The PKC inhibition with chelerythrine chloride (5 mg/kg intraperitoneally) attenuated Hx-induced hippocampal vascular permeability and claudin-5, PKC (h and f) expression, and phosphorylation. This study supports the hypothesis that nPKC-h and aPKC-f signaling mediates TJ protein disruption resulting in increased BBB permeability.

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“…Caco-2 monolayers express the classic PKC isoforms ␣, ␤I and ␤II, ␦, and (34). Dodane and Kachar (35) reported that PKC was the only isoform that colocalized with ZO-1 at tight junction of Madin-Darby canine kidney and Caco-2 cells. However, colonocyte crypt cells of rat and mouse express PKC isoforms ␣, ␤II, ␦, and ⑀ predominantly in the cytosolic compartment (36).…”

Section: Discussionmentioning

confidence: 99%

Protein Kinase C Signaling Regulates ZO-1 Translocation and Increased Paracellular Flux of T84 Colonocytes Exposed toClostridium difficile Toxin A

Chen

Pothoulakis

LaMont

2002

Journal of Biological Chemistry

155

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Clostridium difficile is an anaerobic pathogen that causes antibiotic-associated diarrhea and colitis in humans (1). Two large molecular weight proteins, toxins A and B, secreted by this microbe are glucosyltransferases with substrate specificity for Rho family GTPases (Rho, Rac, and Cdc42) (2, 3). Rho proteins belong to the Ras superfamily of GTPases that regulate cell morphology, gene transcription, and cell proliferation (4). Rho glucosylation by toxins reduces the binding affinity of Rho to its downstream effectors, resulting in inhibition of Rho signaling, disassembly of actin filaments, and cell rounding (5). However, recent studies indicate that C. difficile toxins induce early cellular responses including activation of mitogen-activated protein kinases (6), generation of reactive oxygen metabolites (7), and induction of calcium influx (8) that occur prior to measurable Rho glucosylation, indicating that these and perhaps other Rho-independent signaling events are also involved in toxicity.Dysfunction of paracellular permeability is a major contributor to the increased fluid secretion and diarrhea in C. difficile infection and other inflammatory bowel diseases (9 -11). Tight junctions (TJs), 1 localized in the uppermost basolateral surface of polarized enterocytes, serve as a barrier regulating selective passage of fluid, ions, and lipids through the paracellular pathway (12, 13). Tight junction fibrils are composed of the transmembrane proteins occludin, claudins, and the junctional adhesion molecule whose cytosolic domains interact with the peripheral junctional proteins of the zonula occludens family (ZO-1, -2, and -3) (13). The C-terminal domains of ZOs, in turn, bind to perijunctional actomyosin filaments that support the TJ complex (14). TJ assembly and permeability are regulated by a network of signaling pathways including protein kinase C (PKC), heterotrimeric G proteins, Ras and Rho GTP-binding proteins, protein kinase A, tyrosine kinase, and calcium (15). For example, decreased transepithelial electrical resistance (TER) and increased paracellular flux are observed in cells exposed to the PKC agonist (12-O-tetradecanoylphorbol-13-acetate) (16). Both PKC␣ and -␦ are implicated in the PKC-dependent regulation of TJ assembly and barrier function following activation of a G-protein-coupled receptor (17)(18)(19)(20). Freeze fracture electron micrographs reveal abnormal TJ morphology in transfectants overexpressing mutant forms of the Rho family of GTPases, suggesting involvement of Rho proteins in TJ assembly (21). In polarized intestinal epithelial cells (T84 cells) infected with a chimeric protein containing Clostridium botulinum C3 exoenzyme that specifically inactivates RhoA, perijunctional actin rings are disrupted, leading to a decrease in TER and redistribution of .The increase in TJ permeability and redistribution of TJ proteins in T84 cells exposed to C. difficile toxins has been correlated with depolymerization of the actin cytoskeleton (23,24). However, we previously observed a rapid drop ...

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Identification of Isoforms of G Proteins and PKC that Colocalize with Tight Junctions

Cited by 119 publications

References 35 publications

Gα12 Directly Interacts with PP2A

Gα12 Directly Interacts with PP2A

Protein Kinase C Activation Modulates Reversible Increase in Cortical Blood–Brain Barrier Permeability and Tight Junction Protein Expression during Hypoxia and Posthypoxic Reoxygenation

Protein Kinase C Signaling Regulates ZO-1 Translocation and Increased Paracellular Flux of T84 Colonocytes Exposed toClostridium difficile Toxin A

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