A Key Role for the Phosphorylation of Ser440 by the Cyclic AMP-dependent Protein Kinase in Regulating the Activity of the Src Homology 2 Domain-containing Inositol 5′-Phosphatase (SHIP1)

Abstract

). Using a combination of approaches, we identified the serine residue regulating SHIP1 activity. After mass spectrometric identification of 17 serine and threonine residues on SHIP1 as being phosphorylated by PKA in vitro, studies with truncation mutants of SHIP1 narrowed the phosphorylation site to the catalytic region between residues 400 and 866. Of the two candidate phosphorylation sites located in this region (Ser 440 and Ser 2 is central to many intracellular signaling cascades (1). PtdIns-3,4,5-P 3 is produced in the inner leaflet of the plasma membrane by a family of phosphatidylinositol 3-kinases that are activated by receptor tyrosine kinases or G proteincoupled receptors (2). The PtdIns-3,4,5-P 3 localized at the plasma membrane forms a docking site to attract and regulate downstream signaling molecules containing pleckstrin homology domains, such as the serine kinase, Akt (PKB), that has an essential role in stimulating cell proliferation, growth, survival, and metabolism (2, 3). The level of PtdIns-3,4,5-P 3 in the membrane is opposed by two inositol lipid phosphatases, PTEN (phosphatase and tensin homologue) and SHIP (SH2 domaincontaining inositol 5Ј-phosphatase) (4). SHIP opposes the effects of phosphatidylinositol 3-kinases by dephosphorylating the 5Ј-position on the inositol ring of PtdIns-3,4,5-P 3 and producing phophatidylinositol 3,4-bisphosphate (5).There are two major members of the SHIP family, SHIP1 and SHIP2, that share a highly homologous catalytic region with less similarity in their C-terminal regions (6). SHIP2 is widely expressed, whereas SHIP1 is only expressed in hematopoietic cells (7). SHIP1 was originally identified based on its ability to bind to cytoplasmic adaptor proteins, such as Shc, Dab-1, and Dok-3 (8). SHIP1 contains an N-terminal SH2 domain that leads to interactions with immune receptors, such as Fc␥RIIB and Fc⑀RI (9), a central inositol 5Ј-phosphatase domain, and two tyrosines within NPXY motifs in the C-terminal prolinerich region (10). Genetic manipulations of SHIP1 in mice have shown that SHIP1 plays important roles in functions of myeloid cells and B lymphocytes; for example, in mature neutrophils and mast cells, PtdIns-3,4,5-P 3 levels and the phosphorylation of Akt activity are significantly elevated in the absence of SHIP1 (11). Similarly, B lymphocytes lacking SHIP1 are insensitive to inhibitory signaling via the Fc␥RIIB receptor (9). Moreover, SHIP1 Ϫ/Ϫ mice have a decreased viability due to an increased infiltration of myeloid cells into the lungs, perhaps due to activation of migration pathways because of the elevated levels of PtdIns-3,4,5-P 3 (12).Due to its importance in hematopoietic cells, the activity of SHIP1 is tightly regulated. One model for regulation of SHIP1 function envisions translocation of SHIP1 from the cytosol to the membrane (13). Upon stimulation by growth factors, cytokine receptors, or specific signaling receptors on lymphocytes, SHIP1 is recruited via its N-terminal SH2 domain to phosphorylated tyrosine residues in receptor kina...

Regulation of alveolar macrophage p40phox: hierarchy of activating kinases and their inhibition by PGE2

“…However, our data indicate that although it may participate, PTEN is not the only phosphatase mediating Akt inhibition because the decrease in stimulated H 2 O 2 elicited by PGE 2 and PKA-RII was minimally abrogated by a PTEN inhibitor (not shown). Alternatively, a phosphatase other than PTEN might be activated, and in this regard, Zhang et al [80] have described PKA activation of SH2 (Src homology 2)-containing inositol phosphatase-1.…”

Regulation of immune cell signaling by SHIP1: A phosphatase, scaffold protein, and potential therapeutic target

“…). SHIP phosphatase activity can be enhanced by PKA‐mediated phosphorylation at Ser440 or by direct binding of PI(3,4)P 2 to the C2 domain . The latter discovery led to the identification of a family of small molecules that bind to SHIP via the same site in the C2 domain and serve as allosteric activators.…”

Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation

“…Other studies identified a PKA phosphorylation site (Ser440) in SHIP1, which, when phosphorylated, significantly enhances the activity of the enzyme. This could contribute to the well-documented negative effects of cAMP-mobilizing signals on PI 3-kinase signaling in immune cells (1798). Some meroterpenoid compounds are also able to activate SHIP1 and exert strong anti-inflammatory effects that could represent a means of controlling PI 3-kinase downstream effectors in hematopoietic cells (1170,1459,1460 (927).…”