Regulation of the Src Homology 2 Domain-containing Inositol 5′-Phosphatase (SHIP1) by the Cyclic AMP-dependent Protein Kinase

Abstract

Many agents that activate hematopoietic cells use phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P 3 ) to initiate signaling cascades. The SH2 domain-containing inositol 5 phosphatase, SHIP1, regulates hematopoietic cell function by opposing the action of phosphatidylinositol 3-kinase and reducing the levels of PtdIns 3,4,5-P 3 . Activation of the cyclic AMPdependent protein kinase (PKA) also opposes many of the proinflammatory responses of hematopoietic cells. We tested to see whether the activity of SHIP1 was regulated via phosphorylation with PKA. We prepared pure recombinant SHIP1 from HEK-293 cells and found it can be rapidly phosphorylated by PKA to a stoichiometry of 0.6 mol of PO 4 /mol of SHIP1. In Overall, activation of G protein-coupled receptors that raise cyclic AMP cause SHIP1 to be phosphorylated and stimulate its inositol phosphatase activity. These results outline a novel mechanism of SHIP1 regulation. Activation of phosphatidylinositol 3-kinase (PtdIns 3-kinase)2 is central to regulation of multiple cell functions including cell shape changes, cell migration, cell activation, and proliferation (1). PtdIns 3-kinase phosphorylates phosphatidylinositol 4,5-bisphosphate in the inner leaflet of the plasma membrane to generate phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P 3 ) (2). PtdIns 3,4,5-P 3 then activates downstream signaling pathways by interacting with pleckstrin homology domain-containing proteins, such as phosphoinositide-dependent kinase 1 and the serine-threonine kinase Akt (3). The finding of abnormal activation of the PtdIns 3-kinase pathway in cancer cells has led to interest in the development of inhibitors for PtdIns 3-kinase (4).The level of PtdIns 3,4,5-P 3 is stimulated by multiple members of the PtdIns 3-kinase family (2) and is opposed by two phosphatidylinositol phosphatases: the Src homology 2 (SH2) domain-containing inositol 5Ј phosphatase (SHIP) and the 3Ј inositol phosphatase, phosphatase and tensin homolog (PTEN) (5). PTEN removes phosphate from the 3Ј position in the inositol ring of PtdIns 3,4,5-P 3 and converts it to phosphatidylinositol 4,5-bisphosphate (6). PTEN has a C2 domain, a PDZbinding motif, and a N-terminal phosphatidylinositol 4,5-bisphosphate binding motif essential for translocation to the membrane and interaction with other regulatory proteins (7). There are serine and threonine residues in PTEN that have been found to be phosphorylated, but their role in regulating the activity of the enzyme is not clear (8). Mutations in the PTEN protein have been observed in many tumors, suggesting a role for this enzyme in cancer (9).In contrast, SHIP dephosphorylates the 5Ј position on the inositol ring and produces phosphatidylinositol 3,4-bisphosphate (10). There are three isoforms of SHIP: the 145-kDa hematopoietic cell restricted SHIP (also known as SHIP1); the 104-kDa stem cell-restricted SHIP, sSHIP; and the more widely expressed 150-kDa SHIP2 (11). SHIP1 is the major inositol phosphatase regulating PtdIns 3,4,5-P 3 in monocytes, macrophages,...

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)

“…However, it is not clear that this phosphorylation event can regulate SHIP1 activity (14). We have demonstrated that SHIP1 can be phosphorylated and activated by the cyclic AMPdependent protein kinase (PKA) both in vitro and in cells, highlighting a new mode of SHIP1 regulation (18).…”

“…Phosflow Fix Buffer I and Perm Buffer III were purchased from BD Biosciences. The sources of all other reagents have been published (18).…”

“…Truncated SHIP1 Construction and Site-directed Mutagenesis-The cDNA encoding mouse SHIP1 was subcloned as described previously into the pCMVTag2C vector (Stratagene), which adds a FLAG epitope to the N terminus of the expressed protein (18). To allow expression of and one-step purification of truncated regions of the SHIP1 protein, cDNA constructs encoding truncated regions of SHIP1 were generated by PCR and subcloned into the pCMVTag2C vector.…”

“…DT40 cell suspensions (9) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin, streptomycin, 2 mM L-glutamine, 1% (v/v) chicken serum (Sigma), and 50 M 2-mercaptoethanol as described (18 (S774A) were generated for these experiments as follows. Thirty g of linearized cDNA encoding wild type, S440A, or S774A SHIP1 was co-transfected by electroporation at 250 V and 960 microfarads into SHIP-deficient DT40 cells (9) in combination with 3 g of linearized pApuro plasmid DNA to provide a selectable puromycin marker.…”

“…Cell Culture and Transfection-HEK-293 cells were cultured in DMEM supplemented with 10% fetal bovine serum and transfected with the plasmids indicated under "Results" using Lipofectamine 2000 (Invitrogen) at a 1:1 ratio (DNA/Lipofectamine, w/v) as described (18). DT40 cell suspensions (9) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin, streptomycin, 2 mM L-glutamine, 1% (v/v) chicken serum (Sigma), and 50 M 2-mercaptoethanol as described (18 (S774A) were generated for these experiments as follows.…”