MATRIX NONENZYMATIC GLYCOSYLATION LEADS TO ALTERED CELLULAR PHENOTYPEAND INTRACELLULAR TYROSINE PHOSPHORYLATIONS

The effect of matrix nonenzymatic glycosylation on signal transduction and the cellular phenotype was examined, Human microvascular endothel ial cells were plated on control or glycated basement membrane-like ma trix. Cells exhibited a decrease in their ability to adhere and spread on modified matrix. The pattern of intracellular tyrosine phosphoryla tion was examined by Western Immunoblotting; a band with 65 kDa mobili ty exhibited a marked reduction of tyrosine phosphorylation in cells a dherent to modified matrix. Immunoprecipitation experiments provided e vidence that this band is paxillin, a member of focal adhesion protein s. Immunoprecipitation with antibodies against focal adhesion kinase ( pp125(FAK)), the enzyme that is thought to regulate paxillin tyrosine phosphorylation, also demonstrated a reduction in tyrosine phosphoryla tion of pp125(FAK). To confirm these biochemical data, adherent cells were examined for the distribution of paxillin, using immunofluorescen ce microscopy; paxillin was seen in focal points peripherally located in cells on nor mal matrix, but lacked this pattern in cells on modifi ed matrix. Actin filaments were also disorganized in cells plated on m odified matrix. These data suggest that matrix nonenzymatic glycosylat ion can interfere with and potentially alter cellular phenotype and in tracellular signaling.