title: 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 phos pha tidyl ino si tol 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 phos pha tidyl ino si tol 3-kinase and reducing the levels of PtdIns 3,4,5-P(3). Activation of the cyclic AMP-de pend ent protein kinase (PKA) also opposes many of the pro-inflammatory responses of hematopoietic cells. We tested to see whether the activity of SHIP1 was regulated via phos pho ryl a tion with PKA. We prepared pure recombinant SHIP1 from HEK-293 cells and found it can be rapidly phos pho ryl a ted by PKA to a stoichiometry of 0.6 mol of PO(4)/mol of SHIP1. In (32)P-labeled HEK-293 cells transfected with SHIP1, stimulation with Sp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt hydrate (Sp-cAMPS) or activation of the beta-adrenergic receptor increased the phos pho ryl a tion state of SHIP1. Inhibition of protein phosphatase activity with okadaic acid also increased the phos pho ryl a tion of SHIP1. Phosphorylation of SHIP1 in vitro or in cells by PKA increased the 5' phosphatase activity of SHIP1 by 2-3-fold. Elevation of Ca(2+) in DT40 cells in response to B cell receptor cross-linking, an indicator of PtdIns 3,4,5-P(3) levels, was markedly blunted by pretreatment with Sp-cAMPS. This effect was absent in SHIP(-/-) DT40 cells showing that the effect of Sp-cAMPS in DT40 cells is SHIP1-de pend ent. Sp-cAMPS also blunted the ability of the B cell receptor to increase the phos pho ryl a tion of Akt in DT40 and A20 cells. 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.
authors.affiliation: Department of Pharmacology, University of Virginia, Charlottevilles, Virginia 22908, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Scott F
authors.initials: SF
authors.lastname: Walk
authors.forename: Kodi S
authors.initials: KS
authors.lastname: Ravichandran
authors.forename: James C
authors.initials: JC
authors.lastname: Garrison
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D000072181:Inositol Polyphosphate 5-Phosphatases; D007668:Kidney / Q000166:cytology; D016393:Lymphoma, B-Cell / Q000378:metabolism; D051379:Mice; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D054645:Protein Phosphatase 1 / Q000378:metabolism; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013873:Thionucleotides / Q000378:metabolism; D018909:src Homology Domains
chemical_list: D011943:Receptors, Adrenergic, beta; D011994:Recombinant Proteins; D013873:Thionucleotides; C016957:adenosine-3',5'-cyclic phosphorothioate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D054645:Protein Phosphatase 1; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases; C000606430:INPP5D protein, human; C000606433:Inpp5d protein, mouse; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases