The role of protein kinases in regulating sheep erythrocyte k-cl
cotransport.
Flatman, Peter W., Norma C. Adragna, and Peter K. Lauf.
Departments of Physiology and Biophysics, and Pharmacology and
Toxicology, Wright State University, Dayton OH
APStracts 3:0045C, 1996.
K-Cl cotransport in sheep erythrocytes can be activated by treatment
either with A23187 and EDTA to reduce internal ionized Mg ([Mg2+]i)
to submicromolar levels, with staurosporine, a potent kinase
inhibitor, or with N-ethylmaleimide (NEM). Activation by these
manoeuvres is prevented and reversed by genistein (Ki 15 [mu]M),
which inhibits tyrosine kinases (TK). The related glycosidated
compound genistin, which does not inhibit TK, does not inhibit
transport, whereas another TK inhibitor, tyrphostin B46, inhibits
both basal and stimulated transport (Ki 28 [mu]M). Cotransport
activation by NEM is prevented and reversed by the phosphatase
inhibitor, calyculin A, and activation by staurosporine occurs only
if cells contain ATP. Increasing [Mg2+]i inhibits cotransport in the
presence of calyculin A whether or not staurosporine is present as
well. Our work suggests that genistein inhibits cotransport through a
TK and that staurosporine and NEM activate cotransport, probably
through inhibition of other kinases, causing stimulation through
dephosphorylation of a protein (possibly the transporter itself) by a
serine/threonine phosphatase. [Mg2+]i inhibits cotransport by
activating a kinase (K0.5 10 [mu]M) which phosphorylates this
protein.
Received 1 March 1995; accepted in final form 24 January 1996.
APS Manuscript Number C115-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 8 February 96