Erythrocyte membrane mechanical function is regulated by the spectrin-based membrane skeleton composed of ␣-and -spectrin, actin, protein 4.1R (4.1R), and adducin. Post-translational modifications of these proteins have been suggested to modulate membrane mechanical function. Indeed, -spectrin phosphorylation by casein kinase I has been shown to decrease membrane mechanical stability. However, the effects of the phosphorylation of skeletal proteins by protein kinase C (PKC), a serine/threonine kinase, have not been elucidated. In the present study, we explored the functional consequences of the phosphorylation of 4.1R and adducin by PKC. We identified Ser-312 in 4.1R as the PKC phosphorylation site. Using antibodies raised against phosphopeptides of 4.1R and adducin, we documented significant differences in the time course of phosphorylation of adducin and 4.1R by PKC. Although adducin was phosphorylated rapidly by the activation of membrane-bound atypical PKC by phorbol 12-myristate 13-acetate stimulation, there was a significant delay in the phosphorylation of 4.1R because of delayed recruitment of conventional PKC from cytosol to the membrane. This differential time course in the phosphorylation of 4.1R and adducin in conjunction with membrane mechanical stability measurements enabled us to document that, although phosphorylation of adducin by PKC has little effect on membrane mechanical stability, additional phosphorylation of 4.1R results in a marked decrease in membrane mechanical stability. We further showed that the phosphorylation of 4.1R by PKC results in its decreased ability to form a ternary complex with spectrin and actin as well as dissociation of glycophorin C from the membrane skeleton. These findings have enabled us to define a regulatory role for 4.1R phosphorylation in dynamic regulation of red cell membrane properties.
Asymmetric distribution of phospholipids is ubiquitous in the plasma membranes of many eukaryotic cells. The majority of the aminophospholipids are located in the inner leaflet whereas the cholinephospholipids are localized predominantly in the outer leaflet. Several functional roles for asymmetric phospholipid distribution in plasma membranes have been suggested. Disruption of lipid asymmetry creates a procoagulant surface on platelets and serves as a trigger for macrophage recognition of apoptotic cells. Furthermore, the dynamic process of phospholipid translocation regulates important cellular events such as membrane budding and endocytosis. In the present study, we used the red cell membrane as the model system to explore the contribution of phospholipid asymmetry to the maintenance of membrane mechanical properties. We prepared two different types of membranes in terms of their phospholipid distribution, one in which phospholipids were scrambled and the other in which the asymmetric distribution of phospholipids was maintained and quantitated their mechanical properties. We documented that maintenance of asymmetric distribution of phospholipids resulted in improved membrane mechanical stability. The greater difficulty in extracting the spectrinactin complex at low-ionic strength from the membranes with asymmetric phospholipid distribution further suggested the involvement of interactions between aminophospholipids in the inner leaflet and skeletal proteins in modulating mechanical stability of the red cell membrane. These findings have enabled us to document a functional role of lipid asymmetry in regulating membrane material properties.A symmetric distribution of phospholipids is ubiquitous in the plasma membranes of many eukaryotic cells. The majority of the aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), are located in the inner leaflet whereas the cholinephospholipids, phosphatidylcholine and sphingomyelin, are localized predominantly in the outer leaflet (1, 2). At least three distinct activities are involved in the regulation of membrane lipid sidedness. The MgATPdependent aminophospholipid translocase, (also known as flippase or ATPase II; ref. 3), is responsible for localization of PS and PE in the inner leaflet by rapidly translocating them from the outer to inner leaflet against the electrochemical gradient (4-6). PS is a better substrate than PE for the aminophospholipid translocase (7-9), and the stoichiometry between aminophospholipid translocation and ATP hydrolysis is close to one (10). The activity of the aminophospholipid translocase is inhibited by high concentrations of Ca 2ϩ (11)(12)(13), by the pseudosubstrates of P-type ATPase such as vanadate (14), acetyl phosphate, and p-nitrophenyl phosphate (15), and by sulfhydryl reactive reagents such as N-ethylmaleimide (16) and pyridyldithioethylamine (PDA), a specific inhibitor of phospholipid translocase (16)(17)(18)(19). The less specific MgATP-requiring phospholipid translocase, floppase, moves phospholipids f...
The mechanical properties of human erythrocyte membrane are largely regulated by submembranous protein skeleton whose principal components are alpha- and beta-spectrin, actin, protein 4.1, adducin, and dematin. All of these proteins, except for actin, are phosphorylated by various kinases present in the erythrocyte. In vitro studies with purified skeletal proteins and various kinases has shown that while phosphorylation of these proteins can modify some of the binary and ternary protein interactions, it has no effect on certain other interactions between these proteins. Most importantly, at present there is no direct evidence that phosphorylation of skeletal protein(s) alters the function of the intact membrane. To explore this critical issue, we have developed experimental strategies to determine the functional consequences of phosphorylation of beta-spectrin on mechanical properties of intact erythrocyte membrane. We have been able to document that membrane mechanical stability is exquisitely regulated by phosphorylation of beta-spectrin by membrane-bound casein kinase I. Increased phosphorylation of beta-spectrin decreases membrane mechanical stability while decreased phosphorylation increases membrane mechanical stability. Our data for the first time demonstrate that phosphorylation of a skeletal protein in situ can modulate physiological function of native erythrocyte membrane.
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