Effects of overexpression of the SH2-containing inositol phosphatase SHIP on proliferation and apoptosis of erythroid AS-E2 cells

Previous studies have demonstrated that SH2-containing inositol phosphatase (SHIP) is involved in the control of B cell, myeloid cell and macrophage activation and proliferation. The goal of the present study was to examine the role of SHIP during proliferation and apoptosis in cells of the erythroid lineage. Wild-type and catalytically inactive SHIP proteins were overexpressed in the erythropoietin (EPO)-dependent cell line AS-E2. Stable overexpression of catalytically inactive SHIP decreased proliferation and resulted in prolonged activation of the extracellular signal-regulated protein kinases ERK1/2 and protein kinase B (PKB), while wild-type SHIP did not affect EPO-mediated proliferation or phosphorylation of ERK and PKB. When AS-E2 cells were EPO deprived a significant increase in apoptosis was observed in clones overexpressing wild type. Mutational analysis showed that this increase in apoptosis was independent of the enzymatic activity of SHIP. The enhanced apoptosis due to overexpression of SHIP was associated with an increase in caspase-3 and -9 activity, without a distinct effect on caspase-8 activity or mitochondrial depolarization. Moreover, in cells overexpressing SHIP apoptosis could be reduced by a caspase-3 inhibitor. These data demonstrate that in the erythroid cell line AS-E2 overexpression of catalytically inactive SHIP reduced proliferation, while overexpression of wild-type SHIP had no effect. Furthermore, overexpression of SHIP enhanced apoptosis during growth factor deprivation by inducing specific caspase cascades, which are regulated independently of the 5-phosphatase activity of SHIP

SH2-Containing Inositol 5′-Phosphatase SHIP2 Associates with the p130(Cas) Adapter Protein and Regulates Cellular Adhesion and Spreading

In a previous study, we found that the SHIP2 protein became tyrosine phosphorylated and associated with the Shc adapter protein in response to the treatment of cells with growth factors and insulin (T. Habib, J. A. Hejna, R. E. Moses, and S. J. Decker, J. Biol. Chem. 273:18605–18609, 1998). We describe here a novel interaction between SHIP2 and the p130(Cas) adapter protein, a mediator of actin cytoskeleton organization. SHIP2 and p130(Cas) association was detected in anti-SHIP2 immunoprecipitates from several cell types. Reattachment of trypsinized cells stimulated tyrosine phosphorylation of SHIP2 and increased the formation of a complex containing SHIP2 and a faster-migrating tyrosine-phosphorylated form of p130(Cas). The faster-migrating form of p130(Cas) was no longer recognized by antibodies to the amino terminus of p130(Cas) and appeared to be generated through proteolysis. Interaction of the SHIP2 protein with the various forms of p130(Cas) was mediated primarily through the SH2 domain of SHIP2. Immunofluorescence studies indicated that SHIP2 localized to focal contacts and to lamellipodia. Increased adhesion was observed in HeLa cells transiently expressing exogenous WT-SHIP2. These effects were not seen with SHIP2 possessing a mutation in the SH2 domain (R47G). Transfection of a catalytic domain deletion mutant of SHIP2 (ΔRV) inhibited cell spreading. Taken together, our studies suggest an important role for SHIP2 in adhesion and spreading

Erythropoietin, Thrombopoietin and Leptin Receptors: Signal transduction pathways

International audienceErythropoietin (Epo), Thrombopoietin (Tpo) and leptin are hormones with distinct physiological properties. While the first two regulate survival, growth and differentiation of erythroid and megakaryocytic progenitors respectively, leptin is crucial for mammalian body weight regulation[1–3]. Receptors for these ligands have been isolated and well- characterized; they belong to the class I cytokine receptor family that includes most interleukin receptors involved in hematopoiesis as well as those for prolactin and growth hormone (see Chapter 7) which play an important role in metabolism and reproduction [4]. This family of receptors shares structural similarities both in their extracellular and intracellular domains and is characterized by the absence of an intrinsic tyrosine kinase. Nevertheless, ligand binding to this type of receptor induces the tyrosine phosphorylation of many cellular substrates including the receptor itself, leading to the activation of distinct signaling pathways. This review will focus first on the structure and biological properties of Epo, Tpo, leptin and their receptors. The second part will summarize the signal transduction pathways induced by these three ligands and their roles in cell proliferation, differentiation and survival