title: Role of paxillin in metabolic oxidative stress-induced cytoskeletal reorganization: involvement of SAPK signal transduction pathway and PTP-PEST gene expression.
abstract: Previous studies have shown that glucose deprivation-induced cell death is associated with apoptosis, which is characterized by cellular membrane blebbing in multi-drug-resistant human breast carcinoma MCF-7/ADR cells. In this study, we investigated the mechanism of glucose deprivation-induced cytoskeletal reorganization, which is known to be responsible for the morphological alterations. An increase in the formation of focal adhesion and stress fibers was observed during the early period of glucose deprivation (1-2 h). However, a disappearance of focal adhesion complexes and a loss of stress fiber formation along with membrane blebbing were observed when glucose deprivation continued. These alterations were delayed in MCF-7/ADR cells transfected with bcl-2 and completely suppressed by treatment with an antioxidant, N-acetyl-L-cysteine. These results indicated that glucose deprivation-induced oxidative stress caused the cytoskeletal reorganization. The glucose deprivation-induced alteration of cytoskeletal organization was further investigated by studying a modification of paxillin, one of the focal adhesion proteins. Immunoblotting with anti-paxillin antibody showed that the paxillin band shifted from 68 kDa to about 80 kDa during 1-4 h of glucose deprivation. The mobility shift indicated the modification of paxillin. This possibility was further studied by an immunoprecipitation assay with anti-paxillin/anti-phosphotyrosine antibody and phosphoamino acid analysis (PAA). The immunoprecipitation study revealed that the level of tyrosine phosphorylation of paxillin was maintained for 2 h and then markedly decreased without a change in the total level of paxillin. The PAA study showed that paxillin is dephosphorylated on tyrosine concurrent with phosphorylation on serine/threonine. Expression of a dominant-negative mutant of c-Jun NH(2)-terminal kinase (JNK1) suppressed glucose deprivation-induced JNK1 activation, PTP-PEST gene expression, and alteration of paxillin. Taken together, these results suggest that the alteration of the phosphorylation/dephosphorylation of paxillin may be related to the cytoskeletal reorganization and these events are mediated by glucose deprivation-induced oxidative stress and the stress-activated protein kinase signal transduction pathway.
authors.affiliation: Department of Molecular Biology and Research Center for Cell Differentiation, College of Natural Sciences, Seoul National University, Seoul, South Korea.
authors.forename: S H
authors.initials: SH
authors.lastname: Song
authors.forename: K H
authors.initials: KH
authors.lastname: Lee
authors.forename: M S
authors.initials: MS
authors.lastname: Kang
authors.forename: Y J
authors.initials: YJ
authors.lastname: Lee
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D001943:Breast Neoplasms; D015815:Cell Adhesion Molecules / Q000502:physiology; D003598:Cytoskeletal Proteins / Q000502:physiology*; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure*; D004317:Doxorubicin / Q000633:toxicity; D018432:Drug Resistance, Multiple; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005947:Glucose / Q000502:physiology; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D051419:Paxillin; D010750:Phosphoproteins / Q000502:physiology*; D054594:Protein Tyrosine Phosphatase, Non-Receptor Type 12; D017027:Protein Tyrosine Phosphatases / Q000235:genetics*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014407:Tumor Cells, Cultured
chemical_list: D000975:Antioxidants; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D004317:Doxorubicin; D020928:Mitogen-Activated Protein Kinases; C516738:PTPN12 protein, human; D054594:Protein Tyrosine Phosphatase, Non-Receptor Type 12; D017027:Protein Tyrosine Phosphatases; D005947:Glucose; D000111:Acetylcysteine