title: Matrix nonenzymatic glycosylation leads to altered cellular phenotype and intracellular tyrosine phosphorylation.
abstract: The effect of matrix nonenzymatic glycosylation on signal transduction and the cellular phenotype was examined. Human microvascular endothelial cells were plated on control or glycated basement membrane-like matrix. Cells exhibited a decrease in their ability to adhere and spread on modified matrix. The pattern of intracellular tyrosine phosphorylation was examined by Western Immunoblotting; a band with 65 kDa mobility exhibited a marked reduction of tyrosine phosphorylation in cells adherent to modified matrix. Immunoprecipitation experiments provided evidence that this band is paxillin, a member of focal adhesion proteins. Immunoprecipitation with antibodies against focal adhesion kinase (pp125FAK), the enzyme that is thought to regulate paxillin tyrosine phosphorylation, also demonstrated a reduction in tyrosine phosphorylation of pp125FAK. To confirm these biochemical data, adherent cells were examined for the distribution of paxillin, using immunofluorescence microscopy; paxillin was seen in focal points peripherally located in cells on normal matrix, but lacked this pattern in cells on modified matrix. Actin filaments were also disorganized in cells plated on modified matrix. These data suggest that matrix nonenzymatic glycosylation can interfere with and potentially alter cellular phenotype and intracellular signaling.
authors.affiliation: Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis 55455.
authors.forename: G
authors.initials: G
authors.lastname: Hasegawa
authors.forename: A J
authors.initials: AJ
authors.lastname: Hunter
authors.forename: A S
authors.initials: AS
authors.lastname: Charonis
mesh_terms: D000818:Animals; D001485:Basement Membrane / Q000502:physiology*; D015153:Blotting, Western; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D005109:Extracellular Matrix / Q000502:physiology*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008833:Microcirculation; D009374:Neoplasms, Experimental / Q000473:pathology / Q000503:physiopathology; D051419:Paxillin; D010641:Phenotype; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction; D012867:Skin / Q000098:blood supply; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
chemical_list: D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C496051:Ptk2 protein, mouse