Hydrogen peroxide activates focal adhesion kinase and c-Src by a phosphatidylinositol 3 kinase-dependent mechanism and promotes cell migration in Caco-2 cell monolayers

Shyamali Basuroy, Mitzi Dunagan, Parimal Sheth, Ankur Seth, Radhakrishna Rao

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Abstract

Recent studies showed that c-Src and phosphatidylinositol 3 (PI3) kinase mediate the oxidative stress-induced disruption of tight junctions in Caco-2 cell monolayers. The present study evaluated the roles of PI3 kinase and Src kinase in the oxidative stress-induced activation of focal adhesion kinase (FAK) and acceleration of cell migration. Oxidative stress, induced by xanthine and xanthine oxidase system, rapidly increased phosphorylation of FAK on Y397, Y925, and Y577 in the detergent-insoluble and soluble fractions and increased its tyrosine kinase activity. The PI3 kinase inhibitors, wortmannin and LY294002, and the Src kinase inhibitor, 4-amino-5[chlorophyll]-7-[t-butyl] pyrazolo[3- 4-d]pyrimidine, attenuated tyrosine phosphorylation of FAK. Oxidative stress induced phosphorylation of c-Src on Y418 by a PI3 kinase-dependent mechanism, whereas oxidative stress-induced activation of PI3 kinase was independent of Src kinase activity. Hydrogen peroxide accelerated Caco-2 cell migration in a concentration-dependent manner. Promotion of cell migration by hydrogen peroxide was attenuated by LY294002 and PP2. Reduced expression of FAK by siRNA attenuated hydrogen peroxide-induced acceleration of cell migration. The expression of constitutively active c-SrcY527F enhanced cell migration, whereas the expression of dominant negative c-SrcK296R/Y528F attenuated hydrogen peroxide-induced stimulation of cell migration. Oxidative stress-induced activation of c-Src and FAK was associated with a rapid increase in the tyrosine phosphorylation and the levels of paxillin and p130CAS in actin-rich, detergent-insoluble fractions. This study shows that oxidative stress activates FAK and accelerates cell migration in an intestinal epithelium by a PI3 kinase- and Src kinase-dependent mechanism.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume299
Issue number1
DOIs
StatePublished - Jul 1 2010

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Phosphatidylinositol 3-Kinase
Focal Adhesion Protein-Tyrosine Kinases
Caco-2 Cells
Hydrogen Peroxide
Cell Movement
Oxidative Stress
src-Family Kinases
Phosphorylation
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
Detergents
Tyrosine
Paxillin
Xanthine
Xanthine Oxidase
Tight Junctions
Chlorophyll
Intestinal Mucosa
Protein-Tyrosine Kinases
Small Interfering RNA
Actins

All Science Journal Classification (ASJC) codes

  • Gastroenterology
  • Physiology (medical)
  • Physiology
  • Hepatology

Cite this

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title = "Hydrogen peroxide activates focal adhesion kinase and c-Src by a phosphatidylinositol 3 kinase-dependent mechanism and promotes cell migration in Caco-2 cell monolayers",
abstract = "Recent studies showed that c-Src and phosphatidylinositol 3 (PI3) kinase mediate the oxidative stress-induced disruption of tight junctions in Caco-2 cell monolayers. The present study evaluated the roles of PI3 kinase and Src kinase in the oxidative stress-induced activation of focal adhesion kinase (FAK) and acceleration of cell migration. Oxidative stress, induced by xanthine and xanthine oxidase system, rapidly increased phosphorylation of FAK on Y397, Y925, and Y577 in the detergent-insoluble and soluble fractions and increased its tyrosine kinase activity. The PI3 kinase inhibitors, wortmannin and LY294002, and the Src kinase inhibitor, 4-amino-5[chlorophyll]-7-[t-butyl] pyrazolo[3- 4-d]pyrimidine, attenuated tyrosine phosphorylation of FAK. Oxidative stress induced phosphorylation of c-Src on Y418 by a PI3 kinase-dependent mechanism, whereas oxidative stress-induced activation of PI3 kinase was independent of Src kinase activity. Hydrogen peroxide accelerated Caco-2 cell migration in a concentration-dependent manner. Promotion of cell migration by hydrogen peroxide was attenuated by LY294002 and PP2. Reduced expression of FAK by siRNA attenuated hydrogen peroxide-induced acceleration of cell migration. The expression of constitutively active c-SrcY527F enhanced cell migration, whereas the expression of dominant negative c-SrcK296R/Y528F attenuated hydrogen peroxide-induced stimulation of cell migration. Oxidative stress-induced activation of c-Src and FAK was associated with a rapid increase in the tyrosine phosphorylation and the levels of paxillin and p130CAS in actin-rich, detergent-insoluble fractions. This study shows that oxidative stress activates FAK and accelerates cell migration in an intestinal epithelium by a PI3 kinase- and Src kinase-dependent mechanism.",
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AU - Basuroy, Shyamali

AU - Dunagan, Mitzi

AU - Sheth, Parimal

AU - Seth, Ankur

AU - Rao, Radhakrishna

PY - 2010/7/1

Y1 - 2010/7/1

N2 - Recent studies showed that c-Src and phosphatidylinositol 3 (PI3) kinase mediate the oxidative stress-induced disruption of tight junctions in Caco-2 cell monolayers. The present study evaluated the roles of PI3 kinase and Src kinase in the oxidative stress-induced activation of focal adhesion kinase (FAK) and acceleration of cell migration. Oxidative stress, induced by xanthine and xanthine oxidase system, rapidly increased phosphorylation of FAK on Y397, Y925, and Y577 in the detergent-insoluble and soluble fractions and increased its tyrosine kinase activity. The PI3 kinase inhibitors, wortmannin and LY294002, and the Src kinase inhibitor, 4-amino-5[chlorophyll]-7-[t-butyl] pyrazolo[3- 4-d]pyrimidine, attenuated tyrosine phosphorylation of FAK. Oxidative stress induced phosphorylation of c-Src on Y418 by a PI3 kinase-dependent mechanism, whereas oxidative stress-induced activation of PI3 kinase was independent of Src kinase activity. Hydrogen peroxide accelerated Caco-2 cell migration in a concentration-dependent manner. Promotion of cell migration by hydrogen peroxide was attenuated by LY294002 and PP2. Reduced expression of FAK by siRNA attenuated hydrogen peroxide-induced acceleration of cell migration. The expression of constitutively active c-SrcY527F enhanced cell migration, whereas the expression of dominant negative c-SrcK296R/Y528F attenuated hydrogen peroxide-induced stimulation of cell migration. Oxidative stress-induced activation of c-Src and FAK was associated with a rapid increase in the tyrosine phosphorylation and the levels of paxillin and p130CAS in actin-rich, detergent-insoluble fractions. This study shows that oxidative stress activates FAK and accelerates cell migration in an intestinal epithelium by a PI3 kinase- and Src kinase-dependent mechanism.

AB - Recent studies showed that c-Src and phosphatidylinositol 3 (PI3) kinase mediate the oxidative stress-induced disruption of tight junctions in Caco-2 cell monolayers. The present study evaluated the roles of PI3 kinase and Src kinase in the oxidative stress-induced activation of focal adhesion kinase (FAK) and acceleration of cell migration. Oxidative stress, induced by xanthine and xanthine oxidase system, rapidly increased phosphorylation of FAK on Y397, Y925, and Y577 in the detergent-insoluble and soluble fractions and increased its tyrosine kinase activity. The PI3 kinase inhibitors, wortmannin and LY294002, and the Src kinase inhibitor, 4-amino-5[chlorophyll]-7-[t-butyl] pyrazolo[3- 4-d]pyrimidine, attenuated tyrosine phosphorylation of FAK. Oxidative stress induced phosphorylation of c-Src on Y418 by a PI3 kinase-dependent mechanism, whereas oxidative stress-induced activation of PI3 kinase was independent of Src kinase activity. Hydrogen peroxide accelerated Caco-2 cell migration in a concentration-dependent manner. Promotion of cell migration by hydrogen peroxide was attenuated by LY294002 and PP2. Reduced expression of FAK by siRNA attenuated hydrogen peroxide-induced acceleration of cell migration. The expression of constitutively active c-SrcY527F enhanced cell migration, whereas the expression of dominant negative c-SrcK296R/Y528F attenuated hydrogen peroxide-induced stimulation of cell migration. Oxidative stress-induced activation of c-Src and FAK was associated with a rapid increase in the tyrosine phosphorylation and the levels of paxillin and p130CAS in actin-rich, detergent-insoluble fractions. This study shows that oxidative stress activates FAK and accelerates cell migration in an intestinal epithelium by a PI3 kinase- and Src kinase-dependent mechanism.

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