Volume Expansion Stimulates p72 and p56 in Skate Erythrocytes

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 32 P in the kinase immunoprecipitated from cells prelabeled with 32 PO 4 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.

Hypotonicity-induced Exocytosis of the Skate Anion Exchanger skAE1

“…Additionally the level of tyrosine phosphorylation increases (present studies and Ref. 21). However, the interaction of skAE1 with the cytoskeletal proteins and tyrosine kinases such as p72syk as well as the interaction of these proteins with membrane fractions under normal volume as well as expanded conditions is presently only partially understood.…”

“…This may be due to the tyrosine kinase p72syk, which we have previously demonstrated is stimulated by hypotonic volume expansion (21). Additionally skAE1 in the lipid raft fractions that face outside the cell forms tetrameric complexes.…”

“…Pellets were extracted with 10% (w/v) perchloric acid, allowed to sit on ice for at least 15 min, and protein-precipitated (2000 ϫ g for 5 min at room temperature); the supernatant was removed; and 3 H was quantitated by liquid scintillation spectroscopy. Separation of skAE1 in Varying Membrane Domains-Red blood cell membranes were isolated by protocols published previously (21)(22)(23)(24). Briefly red cell ghosts were made by lysing cells in 20 volumes of 10 mM Tris, pH 7.2, 5 mM EDTA with protease inhibitors (Complete inhibitor mixture, Roche Applied Science) and phosphatase inhibitors (0.1 mM sodium orthovanadate, 1 mM NaF).…”

The Src family kinases: distinct functions of c-Src, Yes, and Fyn in the liver

“…For example, hypoosmolarity can activate Src (18) , Lyn (19) , and Lck (20) , whereas hyperosmolarity activates Yes (10) , Fyn (21) , Hck, and Fgr (22) . The underlying mechanisms are cell type-specific and can involve integrin, EGFR, and ion channel activation (2,4,18,23) .…”

Protein-Tyrosine Kinase Syk Expressed in Human Nasal Fibroblasts and Its Effect on RANTES Production

“…Syk, one of the well-known tyrosine kinases, is widely expressed and plays an important role in intracellular signal transduction in hemopoietic cells: B cells (15,16), mast cells (17), platelets (18), macrophages (19), monocytes (20,21), basophils (22), neutrophils (23), T cells (24), NK cells (25), eosinophils (26), and erythrocytes (27). However, there has been no study that demonstrates the expression of Syk in human nonhemopoietic cells.…”