Abstract
Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and β- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1−/− erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1−/− erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes.
Highlights
Erythrocytes are unique cells in that they have a biconcave shape, can repeatedly deform and regain their shape, and can withstand turbulence and shear forces exerted upon them by fine capillary vessel walls [1]
These studies indicate that disruption of Hem1 results in altered erythrocyte morphology, which correlates with disrupted organization of the actin membrane skeleton
The majority of erythrocyte disorders in humans are associated with mutations in genes encoding important structural proteins, which result in disruption of the erythrocyte actin membrane skeleton, decreased deformability, and subsequent removal of erythrocytes from circulation [1]
Summary
Erythrocytes are unique cells in that they have a biconcave shape, can repeatedly deform and regain their shape, and can withstand turbulence and shear forces exerted upon them by fine capillary vessel walls [1] These complimentary features of strength and flexibility are intrinsic to the interplay between their fluid cell membrane and the resilient yet pliable lattice structure of the membrane skeleton [1,2]. The plasma membrane consists of a phospholipid and cholesterol bilayer interspersed with critical transmembrane proteins such as band 3 and glycophorins A and C (see [3] for review). These transmembrane proteins form two major intracellular complexes called the ‘‘Ankyrin Complex’’
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