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

Abstract

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-1alpha, PP-1gamma, PP-1delta 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-1alpha 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-1alpha 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-1alpha activity controlled in turn by Src-family kinases.

Erythrocyte membrane changes of chorea-acanthocytosis are the result of altered Lyn kinase activity

“…Otherwise, these proteins showed reduced Tyr phosphorylation in PP1/PP2-treated ChAc RBCs, suggesting that Lyn might be also part of other signaling pathways involving other kinases or play a role as downstream regulator of phosphatase(s) similarly to what we previously described in other RBC models. 31,32 …”

Oxidation Impacts the Intracellular Signaling Machinery in Hematological Disorders

“…Prx2 might act as an antioxidant and atypical molecular chaperone targeting free heme to control severe oxidation [82,83,106]. Indeed, the beneficial Notably, reticulocytes dismissed from the BM still express SFKs such as Lyn, Fyn, Fgr, or HcK as well as Syk, a Src related kinase [5,6,10,19]. In diseased red cells such as SCD, kinases such as Erk1/2 have been reported to contribute to adhesive events between red cells and inflammatory activated vascular endothelial surface [103,104].…”

“…In addition, ROS might promote the transient oxidation of cysteine groups on proteins involved in signaling networks, contributing to protein conformational changes and affecting cell signaling. This results in either a block of protein function such as in protein tyrosine phosphatase (PTP) or protein phosphatase 1 and 2 (PP1, PP2A), or activation of protein kinases such as Src family kinases (SFKs) [3][4][5]. Furthermore, oxidation might directly induce/modulate kinase activity such as in Src family kinases (SFKs) or Akt serine-threonine kinase [6][7][8][9][10][11].…”

Identification of the WNK-SPAK/OSR1 Signaling Pathway in Rodent and Human Lenses

“…12 In a variety of cell types it has been shown that the activity of KCC and NKCC are reciprocally regulated through changes in their phosphorylation status with phosphorylation activating NKCC and inactivating KCC. [13][14][15][16][17] Recently, the kinases and phosphatases responsible for modulating the KCC and NKCC phosphorylation status have been identified. The key kinases include selected members of the with-no lysine kinase family (WNK 1, 3 and 4), the STE20-related proline alanine-rich kinase (SPAK, or the rat homologue PASK) and the oxidative stress response kinase 1 (OSR1).…”

“…32 In contrast, the protein phosphatases, PP1 and PP2A, have been shown to directly dephosphorylate NKCC and KCC leading to inactivation of NKCC [34][35][36][37] and activation of KCC. 17,31,[38][39][40] PP1 can also indirectly affect the NKCC/KCC phosphorylation by inhibiting SPAK. 27,35 While the phosphatases PP1 and PP2A are known to be actively expressed in rodent and human lenses, [41][42][43][44] less is known about the regulatory kinases.…”