title: Pervanadate activation of intracellular kinases leads to tyrosine phosphorylation and shedding of syndecan-1.
abstract: Syndecan-1 is a transmembrane haparan sulphate proteoglycan that binds extracellular matrices and growth factors, making it a candidate to act between these regulatory molecules and intracellular signalling pathways. It has a highly conserved transmembrane/cytoplasmic domain that contains four conserved tyrosines. One of these is in a consensus sequence for tyrosine kinase phosphorylation. As an initial step to investigating whether or not phosphorylation of these tyrosines is part of a signal-transduction pathway, we have monitored the tyrosine phosphorylation of syndecan-1 by cytoplasmic tyrosine kinases in intact cells. Tyrosine phosphorylation of syndecan-1 is observed when NMuMG cells are treated with sodium orthovanadate or pervanadate, which have been shown to activate intracellular tyrosine kinases. Initial studies with sodium orthovanadate demonstrate a slow accumulation of phosphotyrosine on syndecan-1 over the course of several hours. Pervanadate, a more effective inhibitor of phosphatases, allows detection of phosphotyrosine on syndecan-1 within 5 min, with peak phosphorylation seen by 15 min. Concurrently, in a second process activated by pervanadate, syndecan-1 ectodomain is cleaved and released into the culture medium. Two phosphorylated fragments of syndecan-1 of apparent sizes 6 and 8 kDa remain with the cell after shedding of the ectodomain. The 8 kDa size class appears to be a highly phosphorylated form of the 6 kDa product, as it disappears if samples are dephosphorylated. These fragments contain the C-terminus of syndecan-1 and also retain at least a portion of the transmembrane domain, suggesting that they are produced by a cell surface cleavage event. Thus pervanadate treatment of cells results in two effects of syndecan-1: (i) phosphorylation of one or more of its tyrosines via the action of a cytoplasmic kinase(s) and (ii) cleavage and release of the ectodomain into the medium, producing a C-terminal fragment containing the transmembrane/cytoplasmic domain.
authors.affiliation: Department of Pathology, University of Wisconsin, Madison 53706, USA.
authors.forename: J
authors.initials: J
authors.lastname: Reiland
authors.forename: V L
authors.initials: VL
authors.lastname: Ott
authors.forename: C S
authors.initials: CS
authors.lastname: Lebakken
authors.forename: C
authors.initials: C
authors.lastname: Yeaman
authors.forename: J
authors.initials: J
authors.lastname: McCarthy
authors.forename: A C
authors.initials: AC
authors.lastname: Rapraeger
mesh_terms: D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors*; D011509:Proteoglycans / Q000378:metabolism*; D053668:Syndecan-1; D053667:Syndecans; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology*
chemical_list: D008562:Membrane Glycoproteins; D011509:Proteoglycans; C508242:Sdc1 protein, mouse; D053668:Syndecan-1; D053667:Syndecans; C076715:pervanadate; D014638:Vanadates; D014443:Tyrosine; D006861:Hydrogen Peroxide; D011494:Protein Kinases; D017027:Protein Tyrosine Phosphatases