Galpha12 regulates epithelial cell junctions through Src tyrosine kinases

TY - JOUR

T1 - Galpha12 regulates epithelial cell junctions through Src tyrosine kinases

AU - Meyer, Tobias N

AU - Hunt, Jennifer

AU - Meyer-Schwesinger, Catherine

AU - Denker, Bradley M

PY - 2003/11/1

Y1 - 2003/11/1

N2 - Regulation and assembly of the epithelial cell junctional complex involve multiple signaling mechanisms, including heterotrimeric G proteins. Recently, we demonstrated that Galpha12 binds to the tight junction scaffolding protein ZO-1 through the SH3 domain and that activated Galpha12 increases paracellular permeability in Madin-Darby canine kidney (MDCK) cells (Meyer et al. J Biol Chem 277: 24855-24858, 2002). In the present studies, we explore the effects of Galpha12 expression on tight and adherens junction proteins and examine downstream signaling pathways. By confocal microscopy, we detect disrupted tight and adherens junction proteins with increased actin stress fibers in constitutively active Galpha12 (QLalpha12)-expressing MDCK cells. The normal distribution of ZO-1 and Na-K-ATPase was altered in QLalpha12-expressing MDCK cells, consistent with loss of polarity. We found that the tyrosine kinase inhibitor genistein and the Src-specific inhibitor PP-2 reversibly abrogated the QLalpha12 phenotype on the junctional complex. Junctional protein localization was preserved in PP-2- or genistein-treated QLalpha12-expressing cells, and the increase in paracellular permeability as measured by transepithelial resistance and [3H]mannitol flux was prevented by the inhibitors. Src activity was increased in QLalpha12-expressing MDCK cells as assessed by Src autophosphorylation, and beta-catenin tyrosine phosphorylation was also increased, although there was no detectable increase in Rho activity. Taken together, these results indicate that Galpha12 regulates MDCK cell junctions, in part through Src tyrosine kinase pathways.

AB - Regulation and assembly of the epithelial cell junctional complex involve multiple signaling mechanisms, including heterotrimeric G proteins. Recently, we demonstrated that Galpha12 binds to the tight junction scaffolding protein ZO-1 through the SH3 domain and that activated Galpha12 increases paracellular permeability in Madin-Darby canine kidney (MDCK) cells (Meyer et al. J Biol Chem 277: 24855-24858, 2002). In the present studies, we explore the effects of Galpha12 expression on tight and adherens junction proteins and examine downstream signaling pathways. By confocal microscopy, we detect disrupted tight and adherens junction proteins with increased actin stress fibers in constitutively active Galpha12 (QLalpha12)-expressing MDCK cells. The normal distribution of ZO-1 and Na-K-ATPase was altered in QLalpha12-expressing MDCK cells, consistent with loss of polarity. We found that the tyrosine kinase inhibitor genistein and the Src-specific inhibitor PP-2 reversibly abrogated the QLalpha12 phenotype on the junctional complex. Junctional protein localization was preserved in PP-2- or genistein-treated QLalpha12-expressing cells, and the increase in paracellular permeability as measured by transepithelial resistance and [3H]mannitol flux was prevented by the inhibitors. Src activity was increased in QLalpha12-expressing MDCK cells as assessed by Src autophosphorylation, and beta-catenin tyrosine phosphorylation was also increased, although there was no detectable increase in Rho activity. Taken together, these results indicate that Galpha12 regulates MDCK cell junctions, in part through Src tyrosine kinase pathways.

KW - Adherens Junctions

KW - Animals

KW - Cell Line

KW - Dogs

KW - Epithelial Cells

KW - GTP-Binding Protein alpha Subunits, G12-G13

KW - Intercellular Junctions

KW - src-Family Kinases

U2 - 10.1152/ajpcell.00548.2002

DO - 10.1152/ajpcell.00548.2002

M3 - SCORING: Journal article

C2 - 12890651

VL - 285

SP - C1281-93

JO - AM J PHYSIOL-CELL PH

JF - AM J PHYSIOL-CELL PH

SN - 0363-6143

IS - 5

ER -