Spectrin phosphorylation and shape change of human erythrocyte ghosts.

Abstract

Human erythrocyte membranes in isotonic medium change shape from crenated spheres to biconcave disks and cup-forms when incubated at 37°C in the presence of MgATP (M . P. Sheetz and S. J . Singer, 1977, J. Cell Biol . 73 :638-646) . The postulated relationship between spectrin phosphorylation and shape change (W . Birchmeier and S. J . Singer, 1977, J. Cell Biol . 73 :647-659) is examined in this report .Salt extraction of white ghosts reduced spectrin phosphorylation during shape change by 85-95%. Salt extraction did not alter crenation, rate of MgATP-dependent shape change, or the fraction (>80%) ultimately converted to disks and cup-forms after 1 h. Spectrin was partially dephosphorylated in intact cells by subjection to metabolic depletion in vitro. Membranes from depleted cells exhibited normal shape-change behavior .Shape-change behavior was influenced by the hemolysis buffer and temperature and by the time required for membrane preparation. Tris and phosphate ghosts lost the capacity to change shape after standing for 1-2 h at 0°C. Hemolysis in HEPES or N-tris(hydroxymethyl)methyl -2-aminoethanesulfonic acid yielded ghosts that were converted rapidly to disks in the absence of ATP and did not undergo further conversion to cup-forms. These effects could not be attributed to differential dephsphorylation of spectrin, because dephosphorylation during ghost preparation and incubation was negligible .These results suggest that spectrin phosphorylation is not required for MgATP-dependent shape change . It is proposed that other biochemical events induce membrane curvature changes and that the role of spectrin is passive.The relationship of cell membrane properties to the biconcave shape of normal human erythrocytes has for many years engaged the attention of investigators from several disciplines . Recently, theoretical treatments (11, 16) and "macro" modeling (13) of the membrane have given satisfactory predictions of observed erythrocyte shapes and simple shape transformations . However, no acceptable explanation for these phenomena at the level of membrane molecular organization has yet been advanced . One of the central issues is the role of intracellular ATP. Nakao et al . (41) first described reversible shape transitions associated with changes in erythrocyte ATP content. Weed et al . (59) suggested that the physical basis for the metabolic dependence of both erythrocyte shape and deformability might be ATP-dependent sot-gel transformations at the inner surface of the membrane. The spectrin-actin meshwork beneath the membrane was found to have some properties of an actomyosin-like system (33,34,49,53) and spectrin polypeptide 2 was shown to be phosphorylated (4,29,42,45,46). These findings raised the prospect that actomyosin-like ATPase 430 activity or protein kinase-mediated phosphorylation would prove to be a link between energy metabolism and control of shape in normal erythrocytes (21,30,33,37,43,44,50,53). Isolated erythrocyte membranes also undergo shape transformations induced by ATP (4...

Metabolic remodeling of the human red blood cell membrane

“…One possible candidate is the phosphorylation of the phosphoinositides (PI) because it consumes more ATP than the combined phosphorylation of all the membrane proteins (23). PIP 2 , phosphorylated from PI by ATP, is thought to play an important role in modulating the binding of the lipid bilayer to the cytoskeleton by altering the protein interactions that comprise the junctional complex at spectrin tetramer ends (24). For example, PIP 2 strengthens the binding affinity of protein-4.1 to glycophorin C (GP) (25) (Fig.…”

ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane: relation to shape changes.

“…The ATP requirement for the maintenance of the discoid shape on both erythrocytes and ghosts is well known (21)(22)(23). Hypotheses of a role of spectrin (24) or lipid (25) phosphorylation in mediating this effect have been recently dismissed (26)(27)(28). In our experiments on both cells and ghosts, the presence of internal ATP, in addition to its effect on phospholipid asymmetry, also engenders a discoid shape.…”

Erythrocytic Casein Kinase II Regulates Cytoadherence of Plasmodium falciparum-infected Red Blood Cells

“…Phosphorylation of erythrocyte cytoskeletal proteins alters their binding affinity for each other, which has important implications in cellular function (23,26,30). VARC is known to tether PfEMP1 to the RBC cytoskeleton by interacting with host proteins actin and spectrin and parasite encoded KAHRP.…”

“…This enzyme is a ubiquitously expressed (in all tissues and organisms) and probably constitutively active Ser/Thr kinase that is located in nearly all subcellular compartments (29). The erythrocyte membrane has a distinct pool of CKII, which along with the cytosolic pool phosphorylates most of the cytoskeletal proteins, including spectrin, ankyrin, and adducin (23,26,30). Our bioinformatics analysis to identify the target residues for phosphorylation along with the kinasing data of N-and C-terminal deletion constructs suggests that most CKII target sites on VARC exist in an N-terminal acidic cluster, typical of CKII recognition sites (Fig.…”