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• W1975987716 abstract "The negative regulatory role of the Src homology 2 domain-containing inositol 5-phosphatase (SHIP) has been invoked in a variety of receptor-mediated signaling pathways. In B lymphocytes, co-clustering of antigen receptor surface immunoglobulin with FcγRIIb promotes the negative effects of SHIP, but how SHIP activity is regulated is unknown. To explore this issue, we investigated the effect of SHIP phosphorylation, receptor tyrosine engagement by its Src homology 2 domain, and membrane recruitment of SHIP on its enzymatic activity. We examined two SHIP phosphorylation kinase candidates, Lyn and Syk, and observed that the Src protein-tyrosine kinase, Lyn is far superior to Syk in its ability to phosphorylate SHIP both in vitro and in vivo. However, we found a minimal effect of phosphorylation or receptor tyrosine engagement of SHIP on its enzymatic activity, whereas membrane localization of SHIP significantly reduced cellular phosphatidylinositol 3,4,5-triphosphate levels. Based on our results, we propose that a membrane localization of SHIP is the crucial event in the induction of its phosphatase effects. The negative regulatory role of the Src homology 2 domain-containing inositol 5-phosphatase (SHIP) has been invoked in a variety of receptor-mediated signaling pathways. In B lymphocytes, co-clustering of antigen receptor surface immunoglobulin with FcγRIIb promotes the negative effects of SHIP, but how SHIP activity is regulated is unknown. To explore this issue, we investigated the effect of SHIP phosphorylation, receptor tyrosine engagement by its Src homology 2 domain, and membrane recruitment of SHIP on its enzymatic activity. We examined two SHIP phosphorylation kinase candidates, Lyn and Syk, and observed that the Src protein-tyrosine kinase, Lyn is far superior to Syk in its ability to phosphorylate SHIP both in vitro and in vivo. However, we found a minimal effect of phosphorylation or receptor tyrosine engagement of SHIP on its enzymatic activity, whereas membrane localization of SHIP significantly reduced cellular phosphatidylinositol 3,4,5-triphosphate levels. Based on our results, we propose that a membrane localization of SHIP is the crucial event in the induction of its phosphatase effects. surface immunoglobulin protein-tyrosine kinase immunoreceptor tyrosine-based activation/inhibition motif Src homology 2 phosphatidylinositol 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate Fc receptor for IgG SH2 domain-containing inositol 5-phosphatase 5)P2, phosphatidylinositol 4,5-bisphosphate polyacrylamide gel electrophoresis Clustering of the B cell surface immunoglobulin (sIg)1 antigen receptor by binding of foreign antigen initiates a set of biochemical events termed positive signaling which activate B lymphocytes to proliferate and secrete soluble antigen specific Ig (reviewed in Refs. 1.Coggeshall K.M. Curr. Top. Microbiol. Immunol. 2000; 245: 213-260PubMed Google Scholar, 2.Coggeshall K.M. Curr. Opin. Immunol. 1998; 10: 306-312Crossref PubMed Scopus (125) Google Scholar, 3.Coggeshall K.M. Immunol. Res. 1999; 19: 47-64Crossref PubMed Scopus (10) Google Scholar). The most proximal signaling event is the stimulation of the Src family of protein-tyrosine kinases (PTKs), which phosphorylates tyrosine residues within conserved immunoreceptor tyrosine-based activation motifs (ITAMs), found in receptor-associated proteins (4.Isakov N. J. Leukocyte Biol. 1997; 61: 6-16Crossref PubMed Scopus (100) Google Scholar). Once the tyrosines in the ITAM are phosphorylated, they serve as docking sites for numerous proteins and enzymes containing Src homology 2 (SH2) domains including the PTK Syk and the p85 adapter subunit of phosphatidylinositol 3-kinase (PtdIns 3-kinase; reviewed in Refs. 5.Campbell K.S. Curr. Opin. Immunol. 1999; 11: 256-264Crossref PubMed Scopus (184) Google Scholar and6.Rudd C.E. Cell. 1999; 96: 5-8Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). These activation signals are then propagated through additional tyrosine phosphorylation and protein-protein interactions and result in changes in B cell biology. PtdIns 3-kinase is comprised of a p85 adapter subunit and a p110 catalytic subunit. By catalyzing the phosphorylation of the D-3 position of the inositol ring (7.Hawkins P.T. Welch H. McGregor A. Eguinoa A. Gobert S. Krugmann S. Anderson K. Stokoe D. Stephens L. Biochem. Soc. Trans. 1997; 25: 1147-1151Crossref PubMed Scopus (31) Google Scholar), PtdIns 3-kinase generates phosphatidylinositol 3,4,5-trisphosphate (PtdIns-(3,4,5)P3), which acts as an intracellular mediator for several enzymes. PtdIns-(3,4,5)P3 binds to pleckstrin homology domains of enzymes (8.Musacchio A. Gibson T. Rice P. Thompson J. Saraste M. Trends Biochem. Sci. 1993; 18: 343-348Abstract Full Text PDF PubMed Scopus (486) Google Scholar) such as Akt (9.Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (447) Google Scholar) and Btk (10.Rameh L.E. Arvidsson A. Carraway III, K.L. Couvillon A.D. Rathbun G. Crompton A. VanRenterghem B. Czech M.P. Ravichandran K.S. Burakoff S.J. Wang D.S. Chen C.S. Cantley L.C. J. Biol. Chem. 1997; 272: 22059-22066Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 11.Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I. Driscoll P.C. Waterfield M.D. Panayotou G. EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (494) Google Scholar, 12.Hyvonen M. Saraste M. EMBO J. 1997; 16: 3396-3404Crossref PubMed Scopus (193) Google Scholar), thereby promoting re-localization to the membrane and providing enzyme access to new lipid substrates or regulatory kinases (13.Klippel A. Reinhard C. Kavanaugh W.M. Apell G. Escobedo M.A. Williams L.T. Mol. Cell. Biol. 1996; 16: 4117-4127Crossref PubMed Scopus (417) Google Scholar). In contrast to activating signals generated upon sIg clustering, co-clustering of sIg with the B cell Fc receptor for IgG (FcγRIIb) aborts B cell activation. It has been proposed (14.Sinclair N.R. Lees R.K. Chan P.L. Khan R.H. Immunology. 1970; 19: 105-116PubMed Google Scholar, 15.Sinclair N.R.S. Chan P.L. Adv. Exp. Med. Biol. 1971; 12: 609-615Crossref Google Scholar) that co-clustering of sIg and FcγRIIb occurs late in the humoral immune response to block continued Ig production. We have termed sIg-FcγRIIb co-clustering “negative signaling,” to contrast with positive signaling initiated by sIg clustering alone and that promotes B cell proliferation. Phillips and Parker (16.Phillips N.E. Parker D.C. J. Immunol. 1983; 130: 602-606PubMed Google Scholar, 17.Phillips N.E. Parker D.C. J. Immunol. 1984; 132: 627-632PubMed Google Scholar, 18.Phillips N.E. Parker D.C. J. Immunol. 1985; 134: 2835-2838PubMed Google Scholar) described an in vitro model using a F(ab′)2 fragment and intact anti-Ig reagents of the IgG class to study biochemical events associated with positive and negative signaling. Earlier studies using this model demonstrated phosphorylation of a tyrosine residue contained within a 13-amino acid motif of the FcγRIIb cytoplasmic tail (19.Muta T. Kurosaki T. Misulovin Z. Sanchez M. Nussenzweig M.C. Ravetch J.V. Nature. 1994; 368: 70-73Crossref PubMed Scopus (436) Google Scholar). Additional experiments showed that certain structural features of the FcγRIIb 13-amino acid motif were shared in common with other receptors that likewise conferred an inhibitory function; accordingly, the motif has been termed the immunoreceptor tyrosine-based inhibitory motif (ITIM; reviewed in Ref. 20.Vely F. Vivier E. J. Immunol. 1997; 159: 2075-2077PubMed Google Scholar). Like signaling through the ITAM, the phosphorylated ITIM recruits SH2 domain-containing molecules to carry out negative signaling. The SH2 domain-containing inositol 5-phosphatase, SHIP, was identified as one of several proteins that bind to the tyrosine-phosphorylated ITIM of FcγRIIb (21.Chacko G.W. Tridandapani S. Damen J. Liu L. Krystal G. Coggeshall K.M. J. Immunol. 1996; 157: 2234-2238PubMed Google Scholar, 22.Ono M. Bolland S. Tempst P. Ravetch J.V. Nature. 1996; 383: 263-266Crossref PubMed Scopus (648) Google Scholar). SHIP is a 145-kDa cytosolic protein that contains a single SH2 domain, a central catalytic region, and two tyrosine phosphorylation sites in the C-terminal region (23.Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (566) Google Scholar, 24.Kavanaugh W.M. Pot D.A. Chin S.M. Deuter-Reinhard M. Jefferson A.B. Norris F.A. Masiarz F.R. Cousens L.S. Majerus P.W. Williams L.T. Curr. Biol. 1996; 6: 438-445Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 25.Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold R. Rohrschneider L.R. Genes Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (378) Google Scholar). In addition to the B cell FcγRIIb, SHIP is recruited to and inhibits cellular activation by a variety of other receptors, including numerous cytokine receptors and the mast cell Fcε receptor (reviewed in Refs.26.Liu L. Damen J.E. Ware M. Hughes M. Krystal G. Leukemia (Baltimore). 1997; 11: 181-184Crossref PubMed Scopus (44) Google Scholar and 27.Huber M. Helgason C.D. Damen J.E. Scheid M. Duronio V. Liu L. Ware M.D. Humphries R.K. Krystal G. Prog. Biophys. Mol. Biol. 1999; 71: 423-434Crossref PubMed Scopus (52) Google Scholar). Recently, in the B cell model, we demonstrated that SHIP was tyrosine-phosphorylated to high stoichiometry and associated with Ras adapter protein Shc only upon co-clustering sIg with FcγRIIb (28.Tridandapani S. Chacko G.W. Brocklyn J.R.v. Coggeshall K.M. J. Immunol. 1997; 158: 1125-1132PubMed Google Scholar). These events were because of the direct recruitment of the SH2 domain of SHIP to the phosphorylated cytoplasmic tyrosine residue of the FcγRIIb ITIM and hence induces SHIP recruitment to the B cell plasma membrane (29.Tridandapani S. Kelley T. Pradhan M. Cooney D. Justement L.B. Coggeshall K.M. Mol. Cell. Biol. 1997; 17: 4305-4311Crossref PubMed Google Scholar). Similar findings of SHIP membrane translocation have been made in T cells stimulated via CD28 (30.Edmunds C. Parry R.V. Burgess S.J. Reaves B. Ward S.G. Eur. J. Immunol. 1999; 29: 3507-3515Crossref PubMed Scopus (30) Google Scholar). The kinase-phosphorylating SHIP is unknown in any cellular system. Studies of SHIP enzymatic activity revealed an exclusive preference for the hydrolysis of 3-phosphoinositides. As such, SHIP can reverse the action of PtdIns 3-kinase by consuming the PtdIns 3-kinase products (31.Deuter-Reinhard M. Apell G. Pot D. Klippel A. Williams L.T. Kavanaugh W.M. Mol. Cell. Biol. 1997; 17: 2559-2565Crossref PubMed Scopus (60) Google Scholar). Other experiments revealed that co-clustering of sIg and FcγRIIb leads to a dramatic reduction of cellular PtdIns-(3,4,5)P3 (32.Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z. Lin S. Gout I. Cantley L.C. Rawlings D.J. Kinet J.P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (386) Google Scholar, 33.Hippen K.L. Buhl A.M. D'Ambrosio D. Nakamura K. Persin C. Cambier J.C. Immunity. 1997; 7: 49-58Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) and reduced activity of distal PtdIns-(3,4,5)P3-responsive enzymes such as Btk (32.Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z. Lin S. Gout I. Cantley L.C. Rawlings D.J. Kinet J.P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (386) Google Scholar, 34.Bolland S. Pearse R.N. Kurosaki T. Ravetch J.V. Immunity. 1998; 8: 509-516Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar) and Akt (35.Jacob A. Cooney D. Tridandapani S. Kelley T. Coggeshall K.M. J. Biol. Chem. 1999; 274: 13704-13710Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 36.Aman M.J. Lamkin T.D. Okada H. Kurosaki T. Ravichandran K.S. J. Biol. Chem. 1998; 273: 33922-33928Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Experiments by our lab (35.Jacob A. Cooney D. Tridandapani S. Kelley T. Coggeshall K.M. J. Biol. Chem. 1999; 274: 13704-13710Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and others (37.Gupta N. Scharenberg A.M. Fruman D.A. Cantley L.C. Kinet J.P. Long E.O. J. Biol. Chem. 1999; 274: 7489-7494Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) showed that the reduction of PtdIns-(3,4,5)P3 is not due to inactivation of PtdIns 3-kinase, because we observed no defect in p85 protein association or in membrane translocation of PtdIns 3-kinase. Therefore, stimulation of SHIP enzymatic activity and the resulting hydrolysis of PtdIns-(3,4,5)P3 are most likely the cause of PtdIns-(3,4,5)P3-dependent enzyme inhibition. Despite the fact that SHIP enzymatic activity is induced by various cytokines and immunoreceptors, it is unclear how the enzymatic activity of SHIP is regulated. Because SHIP was highly phosphorylated upon co-clustering sIg with FcγRIIb (21.Chacko G.W. Tridandapani S. Damen J. Liu L. Krystal G. Coggeshall K.M. J. Immunol. 1996; 157: 2234-2238PubMed Google Scholar) and recruited to the plasma membrane through the engagement with the phosphorylated receptors (29.Tridandapani S. Kelley T. Pradhan M. Cooney D. Justement L.B. Coggeshall K.M. Mol. Cell. Biol. 1997; 17: 4305-4311Crossref PubMed Google Scholar,30.Edmunds C. Parry R.V. Burgess S.J. Reaves B. Ward S.G. Eur. J. Immunol. 1999; 29: 3507-3515Crossref PubMed Scopus (30) Google Scholar, 35.Jacob A. Cooney D. Tridandapani S. Kelley T. Coggeshall K.M. J. Biol. Chem. 1999; 274: 13704-13710Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 38.Tridandapani S. Pradhan M. LaDine J.R. Garber S. Anderson C.L. Coggeshall K.M. J. Immunol. 1999; 162: 1408-1414PubMed Google Scholar), we have formulated three distinct hypotheses that can account for the observed increased SHIP enzymatic activity. First, SHIP enzymatic activity may be directly stimulated by tyrosine phosphorylation. Other enzymes such as Vav (39.Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (680) Google Scholar, 40.Han J. Das B. Wei W. Van Aelst L. Mosteller R.D. Khosravi-Far R. Westwick J.K. Der C.J. Broek D. Mol. Cell. Biol. 1997; 17: 1346-1353Crossref PubMed Scopus (276) Google Scholar) or phospholipase Cγ (41.Kim H.K. Kim J.W. Zilberstein A. Margolis B. Kim J.G. Schlessinger J. Rhee S.G. Cell. 1991; 65: 435-441Abstract Full Text PDF PubMed Scopus (446) Google Scholar) respond in this way to tyrosine phosphorylation. Second, the engagement of a phosphorylated ITIM may promote the elevated activity of SHIP. The SHP-1 tyrosine phosphatase, which binds to the same phosphorylated ITIM of FcγRIIb, exhibits an ∼50-fold enhancement of phosphatase activity upon ITIM engagement by its SH2 domain (42.Pei D. Wang J. Walsh C.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1141-1145Crossref PubMed Scopus (128) Google Scholar). Third, SHIP membrane recruitment may lead to induced enzymatic activity. Many enzymes involved in signal transduction including Akt (43.Astoul E. Watton S. Cantrell D. J. Cell Biol. 1999; 145: 1511-1520Crossref PubMed Scopus (115) Google Scholar), and PtdIns 3-kinase (13.Klippel A. Reinhard C. Kavanaugh W.M. Apell G. Escobedo M.A. Williams L.T. Mol. Cell. Biol. 1996; 16: 4117-4127Crossref PubMed Scopus (417) Google Scholar) are activated by changing their subcellular location from the cytosol to the membrane. To investigate the effect of phosphorylation on SHIP enzymatic activity, we sought the identity of the kinase capable of SHIP phosphorylation. We considered two candidate PTKs, Src family kinases (44.Burg D.L. Furlong M.T. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1994; 269: 28136-28142Abstract Full Text PDF PubMed Google Scholar) and Syk (45.Hutchcroft J.E. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1992; 267: 8613-8619Abstract Full Text PDF PubMed Google Scholar), because members of these two PTK families are present and associated with the ITAMs of sIg and thus are capable of promoting SHIP phosphorylation upon sIg-FcγRIIb co-clustering. We observed that Src family PTKs are superior to Syk regarding SHIP phosphorylation, both in vitro and in vivo. Next, we addressed the three proposed mechanisms of SHIP enzymatic regulation and found a minimal effect on SHIP 5-phosphatase activity by either phosphorylation or engagement of the phosphorylated ITIM of FcγRIIb by the SH2 domain of SHIP. However, enforced plasma membrane localization of SHIP decreased the total amount of cellular PtdIns-(3,4,5)P3 in cells expressing a chimera of human CD8-SHIP, indicating that the subcellular localization directly contributes to SHIP-mediated substrate hydrolysis. Based on these results, we propose that SHIP negatively affects PtdIns-(3,4,5)P3-dependent enzymes by altering its subcellular location from cytosol to the plasma membrane, which is accomplished by recruitment to phosphorylated cytoplasmic tyrosines of receptors. Anti-Lyn and anti-Syk antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antiphosphotyrosine (4G10) monoclonal antibody was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-p85 antisera and anti-SHIP antibody were used as described elsewhere (28.Tridandapani S. Chacko G.W. Brocklyn J.R.v. Coggeshall K.M. J. Immunol. 1997; 158: 1125-1132PubMed Google Scholar, 35.Jacob A. Cooney D. Tridandapani S. Kelley T. Coggeshall K.M. J. Biol. Chem. 1999; 274: 13704-13710Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). PtdIns-(4,5)P2 and PtdIns-(4)P, PP2, and piceatannol were purchased from Calbiochem. Phosphatidylserine and phosphoinositides were obtained from Sigma. Stimulation conditions and other antibodies were described elsewhere (38.Tridandapani S. Pradhan M. LaDine J.R. Garber S. Anderson C.L. Coggeshall K.M. J. Immunol. 1999; 162: 1408-1414PubMed Google Scholar). SHIP cDNA was obtained from Dr. G. Krystal (University of British Columbia), and theEcoRI-PvuII fragment was subcloned into the yeast expression vector, pPICZB-His (Invitrogen, San Diego, CA). The subcloning removes a region encoding the C-terminal 34 amino acids of SHIP; this region contains no phosphorylation site or protein interaction domain and is not part of the catalytic region. Human cDNA encoding CD8α was obtained from Dr. J. Parnes (Stanford University, Palo Alto, CA) and inserted into the EcoRI site of pEF-SHIP (35.Jacob A. Cooney D. Tridandapani S. Kelley T. Coggeshall K.M. J. Biol. Chem. 1999; 274: 13704-13710Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), containing the full-length SHIP cDNA. The resulting material encodes the extracellular and transmembrane domain of CD8α and SHIP as the intracellular domain of the chimera. cDNA encoding murine Lyn was obtained from Dr. A. DeFranco (University of California, San Francisco); cDNA encoding Syk was obtained from Dr. K. Zoller (Ariad Pharmaceuticals, Cambridge, MA). Cloned pPICZB-SHIP-His was transformed to yeast pichia strain, GS115. Transformants expressing high levels of SHIP protein were selected and induced by methanol according to manufacturer's directions. His-tagged SHIP protein was purified by Ni2+-agarose affinity chromatography and subsequent size exclusion chromatography. The resulting material produced a single band upon SDS-PAGE of ∼145 kDa, the expected size of the recombinant protein. To produce the substrate and standards for SHIP 5-phosphatase assay, we prepared [32P]PtdIns-(3,4,5)P3 and [32P]PtdIns-(3,4)P2 essentially as described elsewhere (25.Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold R. Rohrschneider L.R. Genes Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (378) Google Scholar), using commercial sources of PtdIns-(4,5)P2and PtdIns-(4)P (Calbiochem), phosphatidylserine, [γ32P]ATP, and immunoprecipitated PtdIns 3-kinase. All procedures were slightly modified from previous reports (25.Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold R. Rohrschneider L.R. Genes Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (378) Google Scholar). Briefly, 1 × 106cpm of [32P]PtdIns-(3,4,5)P3 in chloroform/methanol (1:1, vol/vol) were evaporated under vacuum and resuspended by sonication in 300 μl of SHIP assay buffer (50 mm HEPES (pH 7.25), 10 mm MgCl2, 1%Nonidet P-40). For 5-phosphatse assay, 25 μl of substrate in SHIP assay buffer, 0.1 mg/ml bovine serum albumin, and various amounts of recombinant SHIP enzyme were mixed to 30 μl of total volume. The reaction was stopped after 20 min at 37 °C by extraction of phospholipids with 100 μl of chloroform/methanol (1:1) and 100 μl of 2 m KCl. The organic phase containing SHIP substrates and products was washed four times with 100 μl of chloroform; the washes and organic phase were combined and evaporated under vacuum. The dried phospholipids were dissolved in 30 μl of chloroform/methanol (1:1), and the material was separated by thin layer chromatography (TLC) using Silica gel 60 plates saturated with 1% potassium oxalate in 50% ethanol, as described earlier (25.Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold R. Rohrschneider L.R. Genes Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (378) Google Scholar). The identity of PtdIns-(3,4,5)P3 and PtdIns-(3,4)P2 were confirmed by comparison of the mobility of [32P]PtdIns-(3,4,5)P3 and [32P]PtdIns-(3,4)P2 prepared separately using authentic commercial standards on the same TLC plate. PtdIns-(3,4,5)P3 and PtdIns-(3,4)P2 were quantified by a Molecular Dynamics Storm system. Lyn from pervanadate (1 mm sodium orthovanadate, 0.6% H2O2)-stimulated A20 B cells and Syk from pervanadate-stimulated THP-1 monocytic cells were immunoprecipitated as described earlier (46.Sarkar S. Schlottmann K. Cooney D. Coggeshall K.M. J. Biol. Chem. 1996; 271: 20182-20186Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) and resuspended in kinase buffer (20 mm HEPES (pH 7.4), 10 mm MgCl2, 5 mm MnCl2). To each reaction, 10 μm cold ATP, 2–4 μCi of [γ-32P]ATP (3000 Ci/mmol), and 2 μg of recombinant SHIP were incubated with the immunoprecipitated kinases for 10 min at 30 °C in a total volume of 25 μl to permit SHIP phosphorylation. The reaction was stopped by adding 5× SDS sample buffer (0.6 m Tris (pH 6.8), 50% glycerol, 12% SDS) and incubating the samples at 95 °C for 5 min. The phosphorylated products, including the autophosphorylated kinases themselves, were analyzed by 7.5% SDS-PAGE, identified by autoradiography, and quantified by a Molecular Dynamics Storm system. To assess the effect of phosphorylation on enzymatic activity, thein vitro kinase reaction was performed as above. Because the Mn2+ cation was inhibitory toward SHIP 5-phosphatase activity (see “Results”), we lowered [Mn2+] by mixing 23 μl of supernatant containing recombinant, phosphorylated SHIP with 1 and 10 mm EDTA and MgCl2, respectively. After incubation on ice for 10 min, 0.1 μg of recombinant SHIP was subjected to 5-phosphatase assay. For transfection into COS-7, 10 μg of cDNA encoding Lyn or Syk PTKs were co-transfected with 10 μg of cDNA encoding SHIP. Cells were harvested after 48 h, stimulated with pervanadate, and analyzed by antiphosphotyrosine blotting as described (21.Chacko G.W. Tridandapani S. Damen J. Liu L. Krystal G. Coggeshall K.M. J. Immunol. 1996; 157: 2234-2238PubMed Google Scholar). In parallel, Lyn and Syk were immunoprecipitated from the SHIP co-transfected cells and assessed forin vitro kinase activity by autophosphorylation and by antiphosphotyrosine blotting. For measurements of SHIP 5-phosphatase activity after tyrosine phosphorylation in vivo, COS-7 cells co-transfected with SHIP and/or Lyn were stimulated with or without pervanadate for 5 min and immunoprecipitated with anti-SHIP antibody. For measurements of SHIP 5-phosphatase activity after stimulation of B cells, lysates of resting or intact anti-Ig-stimulated A20 B cells were incubated with 2.4 μm biotinylated, phosphorylated ITIM peptide, as earlier described (29.Tridandapani S. Kelley T. Pradhan M. Cooney D. Justement L.B. Coggeshall K.M. Mol. Cell. Biol. 1997; 17: 4305-4311Crossref PubMed Google Scholar). The bound material was eluted by incubating streptavidin beads with 100 mm phenylphosphate in SHIP assay buffer, and the eluted material was applied to 5-phosphatase assays. Approximately 9 × 106 COS-7 cells were transiently transfected with pEFneo, pEFneo-SHIP, or pEFneo-CD8-SHIP using LipofectAMINE (Life Technologies, Inc.). 30 h after transfection, cells were labeled to equilibrium by a 16-h incubation with 1 mCi of [32P]orthophosphate in phosphate-free Dulbecco's modified Eagle's medium containing 10% fetal bovine serum exhaustively dialyzed against saline containing 20 mm Hepes. Cells were washed three times in phosphate-free Dulbecco's modified Eagle's medium containing 20 mmHepes, but without 10% fetal bovine serum, resuspended in 100 μl of same buffer, and stimulated with pervanadate for 3 min. Lipid extraction and TLC were performed essentially as described earlier (47.Traynor-Kaplan A.E. Thompson B.L. Harris A.L. Taylor P. Omann G.M. Sklar L.A. J. Biol. Chem. 1989; 264: 15668-15673Abstract Full Text PDF PubMed Google Scholar). [32P]PtdIns-(3,4,5)P3 and [32P]PtdIns-(3,4)P2 were prepared from a PtdIns 3-kinase reaction as described above and used as standards. COS-7 cells were transfected with cDNA encoding wild-type SHIP or the CD8-SHIP chimera. 48 h after transfection, the viable cells were detached by cell dissociation solutionTM (Sigma) and stained with fluorescein isothiocyanate-conjugated anti-CD8 and biotinylated cholera toxin B subunit (to visualize the plasma membrane), followed by Alexa 568 (Molecular Probes, Eugene, OR)-conjugated streptavidin. Additionally, transfected COS-7 cells were permeabilized to identify intracellular proteins with 0.5% Triton X-100 in Tris-buffered saline and immunostained with Alexa 568-conjugated anti-SHIP antibodies. Cells were analyzed by confocal microscopy, and digital images were prepared. Purified SHIP enzyme was prepared from yeast transformed with cDNA encoding SHIP and containing a 6x Histidine tag at the C terminus. About 1 mg of SHIP enzyme was purified from yeast lysates derived from a 1-liter culture (Fig.1 A). We tested the purified recombinant enzyme in a phosphatase assay, using [32P]PtdIns-(3,4,5)P3 prepared from commercial PtdIns-(4,5)P2 and immunoprecipitated PtdIns 3-kinase. The results, shown in Fig. 1 B, indicated that SHIP generated [32P]PtdIns-(3, 4)P2 from [32P]PtdIns-(3,4,5)P3, indicating that the recombinant enzyme had 5-phosphatase activity, as expected. The enzymatic activity was lost in the presence of EDTA, revealing a need for a divalent cation to support SHIP hydrolytic activity toward [32P]PtdIns-(3,4,5)P3. We examined several divalent cations for their ability to support SHIP activity and found (Fig. 2 A) that only Mg2+ was active in this regard. SHIP enzymatic activity was greatly reduced in the presence of Mn2+ and the oxidant, H2O2 (Fig. 2 B). SHIP displayed a relatively broad pH optimum from pH 6 to pH 8 (Fig. 2 C).Figure 2Enzymology of recombinant SHIP. A,divalent cation requirement for SHIP for its activity. Upper panel, TLC: lane 1, SHIP 5-phosphatase assay using recombinant purified SHIP; lane 2, SHIP + 10 mmEDTA; lane 3, SHIP + 1 mm EDTA + 10 mm Mg2+; lane 4, SHIP + 1 mm EDTA + 10 mm Zn2+; lane 5, SHIP + 1 mm EDTA + 10 mmMn2+; lane 6, SHIP + 1 mm EDTA + 10 mm Ca2+. Lower panel, quantification of SHIP assay shown above, reported as a ratio of product to substrate plus product. B, inhibition of SHIP 5-phosphatase activity by H2O2 and Mn2+. SHIP assay was performed in the presence of 0.5 mmH2O2 and 0.3 mm Mn2+; the data are shown as the ratio of product to substrate plus product.Lane 1, without added enzyme; lane 2, plus SHIP;lane 3, SHIP plus 0.5 mmH2O2; lane 4, no enzyme; lane 5, plus SHIP; lane 6, SHIP plus 0.3 mmMnCl2. C, pH dependence of SHIP 5-phosphatase activity. SHIP assays were performed at the pH indicated on thex axis, otherwise as described under “Experimental Procedures.” Shown are the TLC analyses of the reaction products (upper panel) and quantified activities as the ratio of product to substrate plus product (lower panel). These results are representative of five separate and similar assays.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate whether tyrosine phosphorylation of SHIP affects its enzymatic activity, it was necessary to identify the PTK capable of phosphorylating SHIP. Both Src family PTKs and Syk associate with the B cell antigen receptor and either kinase family member are thus properly positioned for carrying out SHIP phosphorylation upon sIg-FcγRIIb co-clustering. Accordingly, we investigated the ability of each of these candidate PTKs to phosphorylate SHIP. Using immunoprecipitated Lyn (as a representative Src family kinase member) or Syk in an in vitro kinase reaction with the recombinant SHIP prepared as described above, we found (Fig. 3 A) that Lyn was far superior to Syk in SHIP phosphorylation; indeed, there was no detectable increase in SHIP tyrosine phosphorylation in the presence of Syk, despite the fact that Syk displayed high autophosphorylating activity (Fig. 3 A,inset 2A summary of the findings appears here; the original data were provided for review. ). As a second approach, we applied the Src family PTK inhibitor PP2 (48.Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1784) Google Scholar) and the Syk inhibitor piceatannol (49.Oliver J.M. Burg D.L. Wilson B" @default.
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• W1975987716 title "Enzymatic Activity of the Src Homology 2 Domain-containing Inositol Phosphatase Is Regulated by a Plasma Membrane Location" @default.
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