Altered phosphorylation of cytoskeleton proteins in sickle red blood cells: the role of protein kinase C, Rac GTPases, and reactive oxygen species

Abstract

The small Rho GTPases Rac1 and Rac2 regulate actin structures and mediate reactive oxygen species (ROS) production via NADPH oxidase in a variety of cells. We have demonstrated that deficiency of Rac1 and Rac2 GTPases in mice disrupts the normal hexagonal organization of the RBC cytoskeleton and reduces erythrocyte deformability. This is associated with increased phosphorylation of adducin at Ser-724, (corresponding to Ser-726 in human erythrocytes), a domain target of protein kinase C (PKC). PKC phosphorylates adducin and leads to decreased F-actin capping and dissociation of spectrin from actin, implicating a significant role of such phosphorylation in cytoskeletal remodeling. We evaluated adducin phosphorylation in erythrocytes from patients with sickle cell disease and found it consistently increased at Ser-726. In addition, ROS concentration is elevated in sickle erythrocytes by 150-250% compared to erythrocytes from normal control individuals. Here, we review previous studies demonstrating that altered phosphorylation of erythrocyte cytoskeletal proteins and increased ROS production result in disruption of cytoskeleton stability in healthy and sickle cell erythrocytes. We discuss in particular the known and potential roles of protein kinase C and the Rac GTPases in these two processes.

Figure 1

A. A working model for the junctional complex of the RBC cytoskeleton, demonstrating the capping actions of tropomodulin and adducin. Tropomodulin, protein 4.1R, and adducin also mediate spectrin-actin association. B. PKC phosphorylates α-adducin at Ser-726 leading to decreased F-actin capping and dissociation of spectrin from actin.

Figure 2

Phosphorylation profile of RBC cytoskeleton proteins in two different representative patients with HbS (SS) and two controls with HbA (AA), as depicted by immunoblotting with A. anti-phosphoserine/threonine antibody, and B. phosphospecific antibody for adducin (Ser-726). Samples representative of more than ten patients per Hb genotype. Actin and GAPDH are shown as loading controls.

Figure 3

Model of NADPH oxidase activation. In the inactive state, gp91^phox (Nox2, the Nox isoform found in neutrophils) and p22^phox exist in a membrane-associated complex, in intracellular vesicles. The p40^phox, p47^phox, and p67^phox subunits are located in the cytoplasm, as is inactive Rac, associated with GDP and RhoGDI. Activation signals result in translocation of p40^phox, p47^phox, p67^phox, and active Rac-GTP to the cell or phagosomal membrane to form the active NADPH oxidase complex with Nox2 and p22^phox. The active complex transfers electrons from NADPH to molecular oxygen to form superoxide radicals. Part of the activation process is postulated to occur by PKC phosphorylation of p47^phox and p67^phox. PKC phosphorylation of RhoGDI may also mediate its dissociation from Rac and the transition from inactive RacGDP to active RacGTP. The basic process of NADPH oxidase activation is similar in other cell types but may involve other members of the Nox, p40^phox, p47^phox, and p67^phox families of proteins.