Embryonic and hematopoietic stem cells express a novel SH2–containing inositol 5′-phosphatase isoform that partners with the Grb2 adapter protein

Abstract

IntroductionEngagement of receptors on the surface of mammalian cells results in the activation of phosphatidylinositol 3-kinase (PI3K) and phosphorylation of phosphatidylinositols on the cytoplasmic side of the cell membrane. 8,15 The generation of phosphatidylinositol 3,4,5-trisphosphate (PI 3,4,5 P 3 ) by PI3K contributes to the activation of signaling pathways that drive cell proliferation. [4][5][6][7] Removal of the phosphate group from the D5 position of phosphatidylinositols by the SH2-containing inositol 5Ј-phosphatase (SHIP) is now recognized as an important negative feedback mechanism on cell activation in the mammalian hematopoietic compartment. 8,9 SHIP was identified based on its ability to bind Grb2, 10 Shc,11,12 and the Fc␥RIIB receptor 13 and by gene trapping. 14 In vitro assays show that SHIP can remove the 5Ј-phosphate of PI 3,4,5 P 3 or inositol 1,3,4,5-tetrakisphosphate, 10,11 suggesting that SHIP may counteract the activity of PI3K 8,9,15 or prevent the sustained influx of Ca ϩϩ into the cell. 9,16,17 SHIP can also form a complex with Shc in stimulated cells. [17][18][19][20][21] Formation of a SHIP-Shc complex is proposed to prevent the recruitment of the nucleotide exchange factor mSos1 to the membrane, thus preventing the activation of the Ras/mitogenactivated protein kinase (MAPK) pathway. 21,22 The ability of SHIP to convert PI 3,4,5 P 3 to PI 3,4 P 2 suggests that SHIP might also influence the activation of protein kinase B/Akt and thus influence the induction of programmed cell death. 7,23,24 There is increasing genetic evidence that SHIP plays an important role as a negative regulator of cell activation in B-lymphoid cells, [1][2][3]6,16 myeloid cells, 1,3 and mast cells. 25 These genetic studies and biochemical analysis of SHIP phosphorylation status indicate that SHIP responds to a wide variety of signals in the hematopoietic compartment, including cytokines, 18,20,26 antigen, 17,19,20 immune complexes, 13,16,27,28 the Fc portion of immunoglobulin G (IgG) antibodies, 29 and thrombin. 30 These results demonstrate that SHIP is an important regulator of cellular responses in the mature cells of several hematopoietic lineages.Here we describe the cloning and characterization of a novel SHIP isoform, s-SHIP. We show that transcription from an internal site within the SHIP gene promotes the expression of s-SHIP in totipotent embryonic stem (ES) cells and hematopoietic stem cells (HSCs), but not in mature hematopoietic cells. The s-SHIP isoform lacks the SH2 domain found in the previously described SHIP isoform whose expression is restricted to the hematopoietic system. Consistent with this structural difference, s-SHIP does not associate in vivo with the Shc adapter protein, but it does associate with Grb2. We propose that s-SHIP plays a unique signaling role in primitive stem cell populations. Materials and methods Cell cultureThe ES cell line, TL1, was a kind gift of Dr Patricia A. Labosky 31 (University of Pennsylvania, Philadelphia) and was cultured in Dulbecco modified Eagle mediu...

Self-renewal of teratocarcinoma and embryonic stem cells

“…How general such restricted pathway activation is for stem cells is unclear. However, it is interesting that variant SHIP and Gab1 signalling molecules lacking subcellular localization motifs have been identified in haematopoietic and ES cells (Tu et al, 2001;Burdon et al, 2002).…”

Induced SHIP Deficiency Expands Myeloid Regulatory Cells and Abrogates Graft-versus-Host Disease

“…SHIP is primarily expressed in hemopoietic cells (9,10), but it can also be expressed in mouse embryonic fibroblasts (11). The SHIP locus also encodes a stem cell-specific isoform called s-SHIP that lacks the Src homology 2 domain and is expressed by pluripotent stem cells and tissue-specific stem cells (11,12). SHIP's role in signal transduction allows it to regulate cell survival, proliferation, apoptosis, and homeostasis of certain hemopoietic cell types (7,(13)(14)(15)(16), as well as primitive stem cell populations (11,12,17,18).…”

“…The SHIP locus also encodes a stem cell-specific isoform called s-SHIP that lacks the Src homology 2 domain and is expressed by pluripotent stem cells and tissue-specific stem cells (11,12). SHIP's role in signal transduction allows it to regulate cell survival, proliferation, apoptosis, and homeostasis of certain hemopoietic cell types (7,(13)(14)(15)(16), as well as primitive stem cell populations (11,12,17,18). Analysis of SHIP-deficient mice revealed that this protein also has a prominent role in the immune system (7,14,15,19).…”

Reversible Ser/Thr SHIP phosphorylation: A new paradigm in phosphoinositide signalling?

“…This enzyme lacks the SH2 domain of the 145-kDa SHIP1 isoform (present in mature cells and referred to as the complete isoform SHIP1). sSHIP is not tyrosine phosphorylated [34,35]. It is, however, present in a membrane fraction due to its interaction with Grb2.…”