Modulation of Erythrocyte Membrane Mechanical Function by Protein 4.1 Phosphorylation

Abstract

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.

Equilibrium physics breakdown reveals the active nature of red blood cell flickering

“…The validity of previous studies, which used equilibrium theories to extract mechanical information on the RBC membrane, has therefore to be critically checked. The comparison of the dissipative response for the three conditions (zero, partial and full ATP depletion) in figure 2c confirms the strong metabolic dependence of passive membrane properties noted previously 6,12,18,22,23 .…”

“…However, the passive mechanical properties of the RBC membrane themselves depend strongly on ATP, as illustrated by the stiffening of the RBC membrane on starvation [17][18][19]6,15 , that correlates with a sudden transition from discocyte to spiculated echinocyte shapes 20,21 . Therefore a direct and compelling explanation for the decrease of fluctuations observed in ATP-depleted cells is the stiffening of the membrane 22,23 rather than the loss of putative active (that is, non-equilibrium) fluctuations. To make a different assessment of the involvement of metabolic activity in flickering, an attractive approach was used by Tuvia et al 14 : at equilibrium, the mean-square fluctuations amplitude is a thermodynamic variable, which, by definition, cannot depend on the dynamics, and in particular on medium viscosity.…”

“…Biochemists identified several phosphorylation sites directly in the network: at the transmembrane protein complexes 4.1R (refs 23,39,40 ) and ankyrin 41 , which anchor the network in the bilayer, but also at the spectrin chains 22 . These phosphorylations have been associated with a decreased mechanical strength of the membrane 42,23,43,6,15 , but the underlying molecular mechanism remains unclear. Instead of considering one specific process, we argue here that any relevant spectrin phosphorylation should lead to a local decrease of the network shear modulus, as this single parameter characterizes its mechanical properties.…”

Direct Cytoskeleton Forces Cause Membrane Softening in Red Blood Cells

“…This phosphorylation is catalyzed by protein kinase C (PKC), which disassembles the spectrin/actin/4.1 trimer, the essential cytoskeletal complex that determines the mechanical stability of the RBC membrane (12). Indeed, PKC activation is known to lead to a decreased overall stability of the membrane skeleton (12,53,54), a structural effect that is consistent with a measurable increase of the dynamic fluctuations of the RBC membrane, as revealed by Betz et al (7). Therefore, assuming the currently accepted mechanochemical model of the 4.1 nodes (51,53), every unpinning event bears a reaction kicking force that should be stressed on the lipid membrane upon dissociation of a spectrin filament from the junctional complex (14,26).…”

A Possible Role for FRM-1, a C. elegans FERM Family Protein, in Embryonic Development

“…The FA domain of the FERM-FA proteins contains conserved motifs for PKC and PKA (Bains, 2006), suggesting that the FA domain plays a regulatory role. For instance, a previous study showed that phosphorylation of the FA domain of 4.1R by PKC interfered with its ability to form a ternary complex with spectrin and actin, which resulted in reduced membrane stability (Manno et al, 2005). The band 4.1 proteins have also been shown to interact with and regulate the membrane localization of various receptors.…”