Volume expansion stimulates p72(syk) and p56(lyn) in skate erythrocytes

Hypotonic volume expansion of skate erythrocytes rapidly stimulates the tyr osine phosphorylation of band 3, the membrane protein thought to mediate th e osmotically sensitive taurine efflux, Skate erythrocytes possess numerous tyrosine kinases including p59fyn, p56lyn, pp60(src), and p72(syk), demons trated by immune complex assays measuring autocatalytic kinase activity. In clusion of the cytoplasmic domain of band 3 in this assay showed that only Syk and Lyn can directly phosphorylate the cytoplasmic domain of band 3, Up on cell volume expansion, Syk activity was increased as assessed by three d ifferent assays (immune complex assay measuring autophosphorylation, assay of the level of phosphotyrosine of the immunoprecipitated kinase, and assay of level of P-32 in the kinase immunoprecipitated from cells prelabeled wi th (PO4)-P-32 and then volume-expanded). The tyrosine kinase Lyn was also s timulated by volume expansion, most notably when analyzed by the latter two methods. Volume expansion stimulated a large increase in the ability of Sy k to phosphorylate band 3 at times that coincide with the stimulation of ta urine flux. The stilbene piceatannol inhibited Syk preferentially over Lyn and other tyrosine kinases and inhibited volume-stimulated taurine efflux i n a concentration-dependent manner similar to that for the inhibition of Sy k, Two major phosphorylation peaks were detected in tryptic digests of cdb3 separated by reverse phase HPLC, Edman degradation demonstrated a phosphot yrosine in a YXXL motif. In conclusion, p72(syk) appears to be a strong can didate as a pivotal signal-transducing step in the volume-activated taurine efflux in skate red cells. The level of band-3 phosphorylation may be regu lated, in addition, by a protein-tyrosine phosphatase of the 1B variety.