PERVANADATE ACTIVATION OF INTRACELLULAR KINASES LEADS TO TYROSINE PHOSPHORYLATION AND SHEDDING OF SYNDECAN-1

Syndecan-1 is a transmembrane haparan sulphate proteoglycan that binds extracellular matrices and growth factors, making it a candidate to a ct between these regulatory molecules and intracellular signalling pat hways. It has a highly conserved transmembrane/cytoplasmic domain that contains four conserved tyrosines. One of these is in a consensus seq uence for tyrosine kinase phosphorylation. As an initial step to inves tigating whether or not phosphorylation of these tyrosines is part of a signal-transduction pathway, we have monitored the tyrosine phosphor ylation of syndecan-1 by cytoplasmic tyrosine kinases in intact cells. Tyrosine phosphorylation of syndecan-1 is observed when NMuMG cells a re treated with sodium orthovanadate or pervanadate, which have been s hown to activate intracellular tyrosine kinases, Initial studies with sodium orthovanadate demonstrate a slow accumulation of phosphotyrosin e on syndecan-1 over the course of several hours. Pervanadate, a more effective inhibitor of phosphatases, allows detection of phosphotyrosi ne on syndecan-1 within 5 min, with peak phosphorylation seen by 15 mi n. Concurrently, in a second process activated by pervanadate, syndeca n-1 ectodomain is cleaved and released into the culture medium. Two ph osphorylated fragments of syndecan-1 of apparent sizes 6 and 8 kDa rem ain with the cell after shedding of the ectodomain. The 8 kDa size cla ss appears to be a highly phosphorylated form of the 6 kDa product, as it disappears if samples are dephosphorylated. These fragments contai n the C-terminus of syndecan-1 and also retain at least a portion of t he transmembrane domain, suggesting that they are produced by a cell s urface cleavage event. Thus pervanadate treatment of cells results in two effects of syndecan-1: (i) phosphorylation of one or more of its t yrosines via the action of a cytoplasmic kinase(s) and (ii) cleavage a nd release of the ectodomain into the medium, producing a C-terminal f ragment containing the transmembrane/cytoplasmic domain.