Regulation of K-Cl cotransport by protein phosphatase 1α in mouse erythrocytes

被引:13
|
作者
Franceschi, L
Villa-Moruzzi, E
Biondani, A
Siciliano, A
Brugnara, C
Alper, S
Lowell, C
Berton, G
机构
[1] Univ Verona, Policlin GB Rossi, Dept Clin Med, Sect Internal Med, I-37134 Verona, Italy
[2] Univ Verona, Policlin GB Rossi, Dept Expt Med, Sect Internal Med, I-37134 Verona, Italy
[3] Univ Pisa, Dept Pathol, I-56100 Pisa, Italy
[4] Harvard Univ, Childrens Hosp, Sch Med, Dept Lab Med, Boston, MA 02115 USA
[5] Harvard Univ, Childrens Hosp, Sch Med, Dept Pathol, Boston, MA 02115 USA
[6] Harvard Univ, Beth Israel Deaconess Med Ctr, Sch Med, Dept Med,Mol & Vasc Med & Renal Unit, Boston, MA 02215 USA
[7] Univ Calif San Francisco, Dept Lab Med, San Francisco, CA 94143 USA
[8] Univ Verona, Dept Pathol, Sect Gen Pathol, I-37100 Verona, Italy
来源
PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY | 2006年 / 451卷 / 06期
关键词
membrane transport; Src-family kinases; PP-1; alpha; membrane oxidative damage; sickle cell disease;
D O I
10.1007/s00424-005-1502-7
中图分类号
Q4 [生理学];
学科分类号
071003 ;
摘要
The K-Cl cotransport (KCC) is an electroneutral-gradient-driven-membrane transport system, which is involved in regulation of red cell volume. Although the regulatory cascade of KCC is largely unknown, a signaling pathway involving phosphatases and kinases has been proposed. Here, we investigated the expression and the activity of protein phosphatase 1(PP-1) isoforms in mouse red cells, focusing on two models of abnormally activated KCC: mice genetically lacking the two Src-family tyrosine kinases, Hck and Fgr, (hck-/-fgr-/-) and the SAD transgenic sickle-cell-mice. The PP-1 alpha, PP-1 gamma, PP-1 delta isoforms were expressed at similar levels in wild-type, hck-/-fgr-/- and SAD mouse erythrocytes and in each case were predominantly localized to cytoplasm. The PP-1 alpha activity was significantly higher in both membrane and cytosol fractions of hck-/-fgr-/- and of SAD erythrocytes than in those of wild-type red cells, suggesting PP-1 alpha as a target of the Hck and Fgr kinases. The PP2, a specific inhibitor of Src-family kinase, significantly increased KCC activity in wild-type mouse red cells, but failed to modify the already increased KCC activity in SAD erythrocytes. The lag-time for activation of KCC was considerably reduced in both hck-/-fgr-/- and SAD erythrocytes, suggesting that the rate limiting activation steps in both strains are freed from their tonic inhibition. Sulfhydryl reduction by dithiothreitol (DTT) lowered KCC activity only in SAD red cells, but did not affect the PP2-treated erythrocytes. These data suggest up-regulation of KCC in SAD red cells is mainly secondary to oxidative damage, which most likely reduces or removes the tonic KCC inhibition resulting from PP-1 alpha activity controlled in turn by Src-family kinases.
引用
收藏
页码:760 / 768
页数:9
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