Most active processes by immune cells including adhesion, migration, and phagocytosis require the coordinated polymerization and depolymerization of filamentous actin (F-actin), which is an essential component of the actin cytoskeleton. This review focuses on a newly characterized hematopoietic cell-specific actin regulatory protein called hematopoietic protein-1 [Hem-1, also known as Nck-associated protein 1-like (Nckap1l or Nap1l)]. Hem-1 is a component of the “WAVE [WASP (Wiskott-Aldrich syndrome protein)-family verprolin homologous protein]” complex, which signals downstream of activated Rac to stimulate F-actin polymerization in response to immuno-receptor signaling. Genetic studies in cell lines and in mice suggest that Hem-1 regulates F-actin polymerization in hematopoietic cells, and may be essential for most active processes dependent on reorganization of the actin cytoskeleton in immune cells.
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.
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Hematopoietic protein-1 (Hem-1) is a hematopoietic cell-specific member of the Hem-family of cytoplasmic adaptor proteins. Orthologs of Hem-1 in flies and worms are essential for cytoskeletal reorganization, embryonic cell migration, and morphogenesis. However, the in vivo functions of mammalian Hem-1 are not known. Using a chemical mutagenesis strategy in mice to identify novel immune function genes, we positionally-cloned a nonsense mutation in the Hem1 gene. Loss of Hem-1 results in defective F-actin polymerization and actin capping in T lymphocytes and neutrophils, due to loss of the Rac GTPase-controlled actin-regulatory "WAVE" complex. T cell development and activation are impaired. Hem1-/- neutrophils are deficient in migration and phagocytic capacity. Conventional "B2" B cell development is inhibited at the pro-B and transitional T1 cell stages, and "B1" B cell development is also impaired. ELISA and ImageStream flow cytometric analyses reveal that NF-¿B-dependent transcription of proinflammatory cytokines, proceed normally in Hem1-/- mice, while TH17 differentiation is enhanced. These results demonstrate that Hem1 is essential for hematopoietic cell development and function by controlling cytoskeletal reorganization.
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