ROLE OF PROTEIN-KINASES IN REGULATING SHEEP ERYTHROCYTE K-CL COTRANSPORT

K-Cl cotransport in sheep erythrocytes can be activated by treatment e ither with A-23187 and EDTA to reduce concentration of internal ionize d Mg ([Mg](i)) to submicromolar levels, with staurosporine, a potent k inase inhibitor, or with N-ethylmaleimide (NEM). Activation by these m aneuvers is prevented and reversed by genistein [inhibition constant ( K-i) of 15 mu M], which inhibits tyrosine kinases (TK). The related gl ycosidated compound genistin, which does not inhibit TK, does not inhi bit transport, whereas another TK inhibitor, tyrphostin B46, inhibits both basal and stimulated transport (K-i of 28 mu M). Cotransport acti vation by NEM is prevented and reversed by the phosphatase inhibitor, calyculin A, and activation by staurosporine occurs only if cells cont ain ATP. Increasing [Mg](i) inhibits cotransport in the presence of ca lyculin A whether or not staurosporine is present as well. Our work su ggests that genistein inhibits cotransport through a TK and that staur osporine and NEM activate cotransport, probably through inhibition of other kinases, causing stimulation through dephosphorylation of a prot ein (possibly the transporter itself) by a serine/threonine phosphatas e. [Mg](i) inhibits cotransport by activating a kinase (concentration for half-maximal activation of 10 mu M) that phosphorylates this prote in.