Abstract
Hypotonic volume expansion of skate erythrocytes rapidly stimulates the tyrosine phosphorylation of band 3, the membrane protein thought to mediate the osmotically sensitive taurine efflux. Skate erythrocytes possess numerous tyrosine kinases including p59fyn, p56lyn, pp60(src), and p72(syk), demonstrated by immune complex assays measuring autocatalytic kinase activity. Inclusion 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. Upon cell volume expansion, Syk activity was increased as assessed by three different assays (immune complex assay measuring autophosphorylation, assay of the level of phosphotyrosine of the immunoprecipitated kinase, and assay of level of 32P in the kinase immunoprecipitated from cells prelabeled with 32PO4 and then volume-expanded). The tyrosine kinase Lyn was also stimulated by volume expansion, most notably when analyzed by the latter two methods. Volume expansion stimulated a large increase in the ability of Syk to phosphorylate band 3 at times that coincide with the stimulation of taurine flux. The stilbene piceatannol inhibited Syk preferentially over Lyn and other tyrosine kinases and inhibited volume-stimulated taurine efflux in a concentration-dependent manner similar to that for the inhibition of Syk. Two major phosphorylation peaks were detected in tryptic digests of cdb3 separated by reverse phase HPLC. Edman degradation demonstrated a phosphotyrosine in a YXXL motif. In conclusion, p72(syk) appears to be a strong candidate as a pivotal signal-transducing step in the volume-activated taurine efflux in skate red cells. The level of band-3 phosphorylation may be regulated, in addition, by a protein-tyrosine phosphatase of the 1B variety.
Highlights
Volume expansion stimulates the efflux of a variety of solutes from cells to accomplish a regulatory volume decrease because of the water that obligatorily follows the solutes
Four tyrosine kinases found in human erythrocytes (Fyn, Lyn, Src, and Syk) and one tyrosine kinase not found in erythrocytes were measured, and their ability to phosphorylate band 3 was determined by immunocomplex kinase assay (Fig. 1)
When three separate experiments were analyzed by densitometry, no significant difference at any time point was band 3 is known to be a prime target for tyrosine phosphorylation by p72syk, we demonstrate the tyrosine phosphorylation of band 3 under physiologically relevant conditions, namely volume expansion
Summary
Volume expansion stimulates the efflux of a variety of solutes from cells to accomplish a regulatory volume decrease because of the water that obligatorily follows the solutes. The formation of this complex is thought to be related to the interaction of band 3 with cytoskeletal proteins in the cell as volume expansion stimulates a high affinity interaction of skate band 3 with ankyrin [7] How this event relates to the formation of the tetramer and how this may mediate taurine efflux are as yet unknown. One biochemical change that occurs in skate band 3 in volume expansion is increased phosphorylation [8] This event occurs very rapidly, and band 3 phosphorylation, that of tyrosine, has been documented in a large number of studies of human red cells The level of band 3 phosphorylation may be regulated by a protein-tyrosine phosphatase type 1B, which is closely associated with band 3
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