HS1 Interacts with Lyn and Is Critical for Erythropoietin-induced Differentiation of Erythroid Cells

Abstract

Erythroid cells terminally differentiate in response to erythropoietin binding its cognate receptor. Previously we have shown that the tyrosine kinase Lyn associates with the erythropoietin receptor and is essential for hemoglobin synthesis in three erythroleukemic cell lines. To understand Lyn signaling events in erythroid cells, the yeast two-hybrid system was used to analyze interactions with other proteins. Here we show that the hemopoietic-specific protein HS1 interacted directly with the SH3 domain of Lyn, via its proline-rich region. A truncated HS1, bearing the Lyn-binding domain, was introduced into J2E erythroleukemic cells to determine the impact upon responsiveness to erythropoietin. Truncated HS1 had a striking effect on the phenotype of the J2E line-the cells were smaller, more basophilic than the parental proerythoblastoid cells and had fewer surface erythropoietin receptors. Moreover, basal and erythropoietin-induced proliferation and differentiation were markedly suppressed. The inability of cells containing the truncated HS1 to differentiate may be a consequence of markedly reduced levels of Lyn and GATA-1. In addition, erythropoietin stimulation of these cells resulted in rapid, endosome-mediated degradation of endogenous HS1. The truncated HS1 also suppressed the development of erythroid colonies from fetal liver cells. These data show that disrupting HS1 has profoundly influenced the ability of erythroid cells to terminally differentiate.

Lyn deficiency reduces GATA-1, EKLF and STAT5, and induces extramedullary stress erythropoiesis

“…Cells were analysed for immunoprecipitation and immunoblotting, essentially as reported previously (Ingley et al, 2000 by lysis in 20 mM Tris-HCl (pH 8.0), 120 mM NaCl, 1.0% Nonident P-40, 10 mM b-glycerophosphate, 10 mM NaF, 1 mM Na 3 VO 4 , 1 mM EDTA, 1 mM EGTA, 10 mM benzamidine, 1 mM PMSF, 10 mg/ml aprotinin. For co-immunoprecipitations, clarified cell lysates were incubated with antibodies to Lyn (SC-15G, SC-15, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or STAT5a/STAT5b (Santa Cruz Biotechno-logy Inc., SC-1081, SC-835) for 2 h at 41C, then collected with protein G-Sepharose beads for 16 h before washing and analysis by immunoblotting.…”

SDF1α‐induced interaction of the adapter proteins Nck and HS1 facilitates actin polymerization and migration in T cells

“…Accordingly, HS1 is thought to stabilize existing branched actin filament networks by bridging F-actin with the Arp2/3 complex [18,19]. A proline-rich domain enables binding to SH3 domain-containing proteins including the Src kinases Lck [20] and Lyn [21]. An SH3 domain forms the C-terminal part of the molecule [3,22].…”

Syk-mediated Tyrosine Phosphorylation Is Required for the Association of Hematopoietic Lineage Cell-specific Protein 1 with Lipid Rafts and B Cell Antigen Receptor Signalosome Complex

“…Direct association of HS1 with Lyn or Lck is evident in T and B lymphocytes and erythroid cells during the differentiation mediated by erythropoietin (21)(22)(23). A complex of HS1 and Lck with human immunodeficiency virus type-1 virions was also reported (24).…”