DEFICIENCY OF SRC FAMILY KINASES FGR AND HCK RESULTS IN ACTIVATION OFERYTHROCYTE K CL COTRANSPORT/

Src-family kinases play a central role in regulation of hematopoietic cell functions. We found that mouse erythrocytes express the Src-famil y kinases Fgr and Hck, as wed as Lyn. To directly test whether Fgr and Hck play any role in erythrocyte function, we analyzed red cells isol ated from fgr(-/-), hck(-/-), and fgr(-/-)hck(-/-) knock-out mice. Mea n corpuscular hemoglobin concentration and median density are increase d, while K content is decreased, in fgr(-/-)hck(-/-) double-mutant ery throcytes compared with wild-type, fgr(-/-), or hck(-/-) erythrocytes. Na/K pump and Na/K/Cl cotransport were not altered, but K/Cl cotransp ort activity was significantly and substantially higher (approximately threefold) in fgr(-/-)hck(-/-) double-mutant erythrocytes. This enhan ced K/Cl cotransport activity did not depend on cell age. In fact, in response to bleeding, K/Cl cotransport activity increased in parallel with reticulocytosis in wild-type erythrocytes, while abnormal K/Cl co transport did not change as a consequence of reticulocytosis in fgr(-/ -)hck(-/-) double-mutant erythrocytes. Okadaic acid, an inhibitor of a phosphatase that has been implicated in activation of the K/Cl cotran sporter, inhibited K/Cl cotransport in wild-type and fgr(-/-)hck(-/-) double-mutant erythrocytes to a comparable extent. In contrast, stauro sporine, an inhibitor of a kinase that has been suggested to negativel y regulate this same phosphatase enhanced K/Cl cotransport in wild-typ e but not in fgr(-/-)hck(-/-) double-mutant erythrocytes. On the basis of these findings, we propose that Fgr and Hck are the kinases involv ed in the negative regulation of the K/Cl cotransporter-activating pho sphatase. Abnormality of erythrocyte K/Cl cotransport in fgr(-/-)hck(- /-) double-mutant animals represents the first demonstration that Src- family kinases may be involved in regulation of membrane transport.