B-cell immune response to antigens is terminated or attenuated by surface receptors such as Fc␥RIIB1 and CD22 on B cells (5,11,34,48). These inhibitory receptors recruit specific intracellular signaling proteins, which play a key role in attenuating the early activation events initiated by cross-linking of the B-cell receptor (BCR). Fc␥RIIB1 is an important mediator of the attenuation of B-cell activation by antibody-antigen immune complexes in the later phases of the immune response (49). Coengagement of Fc␥RIIB1 with BCR results in a potent inhibitory signal that depends on the recruitment of Src homology 2-containing inositol phosphatase (SHIP). SHIP binds to the phosphorylated immunotyrosine-based motif (ITIM) in the cytoplasmic region of Fc␥RIIB1 (43, 44), and SHIP-mediated dephosphorylation of specific phosphoinositide products has been implicated in terminating the BCR-induced activation events (4, 14, 53).SHIP was initially characterized in hematopoietic cells as a 145-kDa phosphoprotein that coprecipitated with the adapter protein Shc upon stimulation of specific receptors (6-8, 37, 50, 52, 54). Molecular cloning of SHIP identified it as a 5Ј-inositolphosphatase (5Ј-IPase), based on homology with other 5Ј-IPases (9,13,29,36,45,57). SHIP specifically dephosphorylates phosphatidylinositol-3,4,5-trisphosphate (PIP3), a major product of phosphoinositide-3-kinase (PI3K) enzymatic action, as well as inositoltetrakisphosphate (IP4), both in vitro (28,36) and in vivo (53). The requirement for SHIP in Fc␥RIIB1-mediated inhibition of BCR signaling has been well established (4,5,14,20,32,44,48,53). Recruitment of enzymatically active SHIP to the receptor complex results in potent inhibition of intracellular calcium flux (12,30,44), diminished activation of the serine-threonine kinase Akt (1, 3, 17, 27), inhibition of the Ras/mitogen-activated protein kinase pathway (56), and the regulation of apoptosis (2, 38, 47). Further evidence for a crucial role for SHIP in negative regulation of BCR signaling comes from studies with SHIP knockout mice as well as SHIP Ϫ/Ϫ Rag Ϫ/Ϫ chimeric mice, in which BCR-mediated responses are heightened and the Fc␥RIIB1-dependent inhibition of BCR responses is abolished (23,39).It is noteworthy that SHIP also negatively regulates histamine release in response to engagement of the immunoglobulin E (IgE) receptor and Steel factor (25,26,43), as well as the proliferative response to interleukin-3 and the macrophage colony-stimulating factor (36). Ex vivo studies with cells from SHIP-deficient mice have suggested that in the absence of SHIP, the myeloid progenitor cells hyperproliferate in response to cytokines and hematopoietic growth factors, with the dose-response curve being left-shifted (23). Taken together, these studies have clearly established a functional role for SHIP as a negative regulator of cytokine and antigen receptor signaling.The 145-kDa isoform of SHIP, the predominant form expressed in hematopoietic cells, is composed of an N-terminal Src homology 2 (SH2) domain, a central...

S-nitrosoglutathione (GSNO) stabilizes the a-subunit of hypoxia inducible factor-1 (HIF-1) in normoxic cells, but not in the presence of PI3K inhibitors. In this report, the biochemical pathway by which GSNO alters PI3K/Akt activity to modify HIF-1 expression was characterized in Cos cells and primary pulmonary vascular endothelial cells. GSNO increased Akt kinase activity-and downstream HIF1a protein accumulation and DNA-binding activity-in a dose-and time-dependent manner. The PI3K inhibitors, wortmannin and LY294002, blocked these responses. Neither glutathione nor 8-bromo-cyclic GMP mimicked the GSNO-induced increases in Akt kinase activity. GSNO-induced Akt kinase activity and downstream HIF-1a stabilization were blocked by acivicin, an inhibitor of g2glutamyl transpeptidase (gGT), a transmembrane protein that can translate extracellular GSNO to intracellular S-nitrosocysteinylglycine. Dithiothreitol blocked GSNO-induced Akt kinase activity and HIF-1a stabilization. Moreover, the 39-phosphatase of phosphoinositides, PTEN (phosphatase and tensin homolog deleted on chromosome ten) was S-nitrosylated by GSNO in pulmonary arterial endothelial cells, which was reversed by dithiothreitol and ultraviolet light. Interestingly, the abundance of S-nitrosylated PTEN also correlated inversely with PTEN activity. Taken together, these results suggest that GSNO induction of Akt appears to be mediated by S-nitrosylation chemistry rather than classic NO signaling through guanylate cyclase/cGMP. We speculate that gGT-dependent activation of Akt and subsequent activation of HIF-1 in vascular beds may be relevant to the regulation of HIF-1-dependent gene expression in conditions associated with oxyhemoglobin deoxygenation, as opposed to profoundly low PO 2 , in the pulmonary vasculature.Keywords: phosphatidylinositol 3-kinase; hypoxia-inducible factor-1; S-nitrosothiol; g-glutamyl transpeptidase; PTEN Hypoxia-inducible factor (HIF)-1 promotes the expression of several genes that confer hypoxic tolerance through angiogenesis, erythropoeisis, vasodilation, and altered glucose metabolism. In hypoxia, the regulatory subunit of the HIF-1 heterodimer, HIF1a, is stabilized through decreased activity of prolyl hydroxylases that target the protein for degradation (1). In normoxia, HIF-1 can also be activated by mitogen-activated protein kinase (MAPK)-dependent processes initiated by growth factors (2-4) or S-nitrosoglutathione (GSNO) (5). Indeed, exposure to GSNO in normoxia results in the accumulation of HIF-1a protein. Of note, inhibitors of the phosphatidylinositol 39-kinase (PI3K)-initiated Akt activation prevent this effect, implicating this signaling pathway in the GSNO effect (6).Redox-associated modification of cysteine thiols by nitric oxide (NO) in biology can regulate the function of proteins (7-9). S-nitrosylated proteins are involved in numerous signaling pathways. Transnitrosation, the transfer of NO in the form of nitrosonium (NO 1 ) from one cysteine thiol to a second cysteine thiol, is believed to be responsible for m...

Activation of the serine/threonine kinase Akt and the regulation of its activation are recognized as critical in controlling proliferative/survival signals via many hematopoietic receptors. In B lymphocytes, the B-cell receptor (BCR)-mediated activation of Akt is attenuated by co–cross-linking of BCR with the inhibitory receptor FcγRIIB1, and the binding of the SH2 domain-containing inositol phosphatase, SHIP, to FcγRIIB1. Because SHIP dephosphorylates phosphatidylinositol 3,4,5-trisphosphate (PIP3) and activation of Akt requires PIP3, the destruction of this phospholipid has been proposed as the mechanism for Akt inhibition. However, upstream kinases that activate Akt, such as PDK1, also require PIP3 for activation. In this report, we addressed whether SHIP inhibits Akt directly at the level of Akt recruitment to the membrane, indirectly through PDK recruitment/phosphorylation of Akt, or both. We generated stable B-cell lines expressing a regulatable, but constitutively membrane-bound Akt that still required PDK-dependent phosphorylation for activation. Several lines of evidence suggested that activation of this membrane-targeted Akt is not inhibited by FcγRIIB1/SHIP and that PDK is not a target for SHIP-mediated inhibition. These data demonstrate that SHIP inhibits Akt primarily through regulation of Akt membrane localization. We also observed during these studies that FcγRIIB1/SHIP does not inhibit p70S6k activation, even though several other PIP3-dependent events were down-regulated. Because the enhanced activation of Akt in the absence of SHIP correlates with hyperproliferation in the myeloid lineage, our data have implications for SHIP and Akt-dependent regulation of proliferation in the hematopoietic lineage.