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• W1993935178 abstract "In human neutrophils, the activation of phospholipase D and the Tyr phosphorylation of proteins are early signaling events upon cell stimulation. We found that the pretreatment of neutrophils with ethanol (0.8%) or 1-butanol (0.3%), which results in the accumulation of phosphatidylalcohol at the expense of phosphatidic acid (PA), decreased the phorbol myristate acetate-stimulated Tyr phosphorylation of endogenous proteins (42, 115 kDa). When neutrophil cytosol was incubated in the presence or absence of PA, these and other endogenous proteins became Tyr-phosphorylated in a PA-dependent manner. In contrast, phosphatidylalcohols exhibited only 25% (phosphatidylethanol) or 5% (phosphatidylbutanol) of the ability of PA to stimulate Tyr phosphorylation in the cell-free assay. Similarly, other phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, polyphosphoinositides, and sphingosine 1-phosphate) showed little ability to stimulate Tyr phosphorylation. These data suggest that PA can function as an intracellular regulator of Tyr phosphorylating activity. Gel filtration chromatography of leukocyte cytosol revealed a peak of PA-dependent Tyr phosphorylating activity distinct from a previously described PA-dependent phosphorylating activity (Waite, K. A., Wallin, R., Qualliotine-Mann, D., and McPhail, L. C. (1997)J. Biol. Chem. 272, 15569–15578). Among the protein Tyr kinases expressed in neutrophils, only Fgr eluted exclusively in the peak of PA-dependent Tyr phosphorylating activity. Importantly, Fgr isolated from unstimulated neutrophil lysates showed increased activity in the presence of PA but not phosphatidylbutanol. Moreover, the pretreatment of neutrophils with 1-butanol decreased Fgr activity in cells stimulated with formyl-methionyl-leucyl phenylalanine plus dihydrocytochalasin B. Together, these results suggest a new second messenger role for PA in the regulation of Tyr phosphorylation. In human neutrophils, the activation of phospholipase D and the Tyr phosphorylation of proteins are early signaling events upon cell stimulation. We found that the pretreatment of neutrophils with ethanol (0.8%) or 1-butanol (0.3%), which results in the accumulation of phosphatidylalcohol at the expense of phosphatidic acid (PA), decreased the phorbol myristate acetate-stimulated Tyr phosphorylation of endogenous proteins (42, 115 kDa). When neutrophil cytosol was incubated in the presence or absence of PA, these and other endogenous proteins became Tyr-phosphorylated in a PA-dependent manner. In contrast, phosphatidylalcohols exhibited only 25% (phosphatidylethanol) or 5% (phosphatidylbutanol) of the ability of PA to stimulate Tyr phosphorylation in the cell-free assay. Similarly, other phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, polyphosphoinositides, and sphingosine 1-phosphate) showed little ability to stimulate Tyr phosphorylation. These data suggest that PA can function as an intracellular regulator of Tyr phosphorylating activity. Gel filtration chromatography of leukocyte cytosol revealed a peak of PA-dependent Tyr phosphorylating activity distinct from a previously described PA-dependent phosphorylating activity (Waite, K. A., Wallin, R., Qualliotine-Mann, D., and McPhail, L. C. (1997)J. Biol. Chem. 272, 15569–15578). Among the protein Tyr kinases expressed in neutrophils, only Fgr eluted exclusively in the peak of PA-dependent Tyr phosphorylating activity. Importantly, Fgr isolated from unstimulated neutrophil lysates showed increased activity in the presence of PA but not phosphatidylbutanol. Moreover, the pretreatment of neutrophils with 1-butanol decreased Fgr activity in cells stimulated with formyl-methionyl-leucyl phenylalanine plus dihydrocytochalasin B. Together, these results suggest a new second messenger role for PA in the regulation of Tyr phosphorylation. 47 kDa ph agocytic ox idase component diisopropyl fluorophosphate, Me2SO, dimethyl sulfoxide formyl-methionyl-leucyl phenylalanine glutathione S-transferase phosphatidic acid phorbol myristate acetate phosphatidylcholine PAGE, polyacrylamide gel electrophoresis Neutrophils are dynamic, short lived cells that play a vital role in the early phases of the immune response. They provide formidable killing mechanisms against invading organisms. Neutrophils sense their environment through a variety of cell surface receptors that allow the cells to respond to bacterial products, inflammatory cytokines, immunoglobulins, complement cleavage products, and adhesion molecules. Upon receptor ligation, numerous signaling events ensue within the cell that culminate in a repertoire of functional responses appropriate for a given stimulus. Among the many signaling events induced, increased Tyr phosphorylation of proteins is commonly observed for a wide variety of neutrophil stimuli (1Rollet E. Caon A.C. Roberge C.J. Liao N.W. Malawista S.E. McColl S.R. Naccache P.H. J. Immunol. 1994; 153: 353-363PubMed Google Scholar, 2Berton G. Curr. Opin. Hematol. 1999; 6: 51-58Crossref PubMed Scopus (27) Google Scholar). Importantly, Tyr phosphorylation appears to play a central role in signaling pathways because treatment of neutrophils with inhibitors of protein Tyr kinases has been shown to interfere with most cellular functions (reviewed in Ref. 3Welch H. Mauran C. Maridonneau-Parini I. Methods Companion Methods Enzymol. 1996; 9: 607-618Crossref Scopus (25) Google Scholar).Neutrophils express a large number of nonreceptor protein Tyr kinases (2Berton G. Curr. Opin. Hematol. 1999; 6: 51-58Crossref PubMed Scopus (27) Google Scholar, 3Welch H. Mauran C. Maridonneau-Parini I. Methods Companion Methods Enzymol. 1996; 9: 607-618Crossref Scopus (25) Google Scholar, 4Brumell J.H. Burkhardt A.L. Bolen J.B. Grinstein S. J. Biol. Chem. 1996; 271: 1455-1461Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) with representatives from eight of the nine known families (5Hardie, G., and Hanks, S., eds. (1995) The Protein Kinase Facts Book, pp. 41-42, Academic Press, LondonGoogle Scholar). The functional roles of only a few of these enzymes in neutrophils have been clearly defined (reviewed in Ref. 2Berton G. Curr. Opin. Hematol. 1999; 6: 51-58Crossref PubMed Scopus (27) Google Scholar). Similarly, little is known about the regulation of protein Tyr kinases in neutrophils. The factors that influence Tyr phosphorylation in neutrophils are diverse and may reflect the variety of enzymes expressed in these cells. Examples of signaling events shown to alter protein Tyr kinase activity include ligand-induced dimerization of the Fc receptor (2Berton G. Curr. Opin. Hematol. 1999; 6: 51-58Crossref PubMed Scopus (27) Google Scholar), reactive oxygen intermediates (4Brumell J.H. Burkhardt A.L. Bolen J.B. Grinstein S. J. Biol. Chem. 1996; 271: 1455-1461Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 6Fialkow L. Kin C.C. Grinstein S. Downey G.P. J. Biol. Chem. 1993; 268: 17131-17137Abstract Full Text PDF PubMed Google Scholar, 7Abe J.-I. Berk B.C. J. Biol. Chem. 1999; 274: 21003-21010Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), and G protein activation (8Nasmith P.E. Mills G.B. Grinstein S. Biochem. J. 1989; 257: 893-897Crossref PubMed Scopus (56) Google Scholar). Recently, the enhancement of Tyr phosphorylation by exogenously added (9Siddiqui R.A. Yang Y.-C. Cell. Signal. 1995; 7: 247-259Crossref PubMed Scopus (33) Google Scholar, 10Siddiqui R.A. English D. Biochim. Biophys. Acta. 1997; 1349: 81-95Crossref PubMed Scopus (42) Google Scholar, 11Ohguchi K. Kasai T. Nozawa Y. Biochim. Biophys. Acta. 1997; 1346: 301-304Crossref PubMed Scopus (20) Google Scholar) or endogenously generated (9Siddiqui R.A. Yang Y.-C. Cell. Signal. 1995; 7: 247-259Crossref PubMed Scopus (33) Google Scholar, 11Ohguchi K. Kasai T. Nozawa Y. Biochim. Biophys. Acta. 1997; 1346: 301-304Crossref PubMed Scopus (20) Google Scholar) PA,1 the lipid product of phospholipase D, has been observed in neutrophils and other cell types. The mechanism by which PA stimulates Tyr phosphorylation has not been addressed.Like Tyr phosphorylation, the activation of phospholipase D is an early cellular signaling event and has been linked to functional responses of the neutrophil (12Bonser R.W. Thompson N.T. Randall R.W. Garland L.G. Biochem. J. 1989; 264: 617-620Crossref PubMed Scopus (166) Google Scholar, 13Lennartz M.R. Int. J. Biochem. Cell Biol. 1999; 31: 415-430Crossref PubMed Scopus (87) Google Scholar). This enzyme metabolizes membrane phospholipids, in a stimulus-dependent manner, yielding PA and the free head group (e.g. choline or ethanolamine). Although slow to gain acceptance as a lipid second messenger, PA has now been reported to interact with or activate a variety of signaling components including protein Ser/Thr kinases (14Limatola C. Schaap D. Moolenaar W.H. van Blitterswijk W.J. Biochem. J. 1994; 304: 1001-1008Crossref PubMed Scopus (284) Google Scholar, 15Ghosh S. Strum J.C. Sciorra V.A. Daniel L.W. Bell R.M. J. Biol. Chem. 1996; 271: 8472-8480Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 16Waite K.A. Wallin R. Qualliotine-Mann D. McPhail L.C. J. Biol. Chem. 1997; 272: 15569-15578Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 17Bokoch G.M. Reilly A.M. Daniels R.H. King C.C. Olivera A. Spiegel S. Knaus U.G. J. Biol. Chem. 1998; 273: 8137-8144Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), protein Ser/Thr (18Kishikawa K. Chalfant C.E. Perry D.K. Bielawska A. Hannun Y.A. J. Biol. Chem. 1999; 274: 21335-21341Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) and Tyr (19Zhao Z. Shen S.-H. Fischer E.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4251-4255Crossref PubMed Scopus (108) Google Scholar) phosphatases, a cyclic nucleotide phosphodiesterase (20Marcoz P. Nemoz G. Prigent A.-F. Lagarde M. Biochim. Biophys. Acta. 1993; 1176: 129-136Crossref PubMed Scopus (31) Google Scholar), regulators of G proteins (21Tsai M.-H., Yu, C.-L. Wei F.-S. Stacey D.W. Science. 1989; 243: 522-526Crossref PubMed Scopus (232) Google Scholar, 22Tsai M.-H. Hall A. Stacey D.W. Mol. Cell. Biol. 1989; 9: 5260-5264Crossref PubMed Scopus (54) Google Scholar), lipid kinases (23Moritz A. De Graan P.N.E. Gispen W.H. Wirtz K.W.A. J. Biol. Chem. 1992; 267: 7207-7210Abstract Full Text PDF PubMed Google Scholar), and phospholipases (24Jones G.A. Carpenter G. J. Biol. Chem. 1993; 268: 20845-20850Abstract Full Text PDF PubMed Google Scholar, 25Geng D. Chura J. Roberts M.F. J. Biol. Chem. 1998; 273: 12195-12202Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Thus it is possible that the PA-stimulated Tyr phosphorylation previously observed (9Siddiqui R.A. Yang Y.-C. Cell. Signal. 1995; 7: 247-259Crossref PubMed Scopus (33) Google Scholar, 10Siddiqui R.A. English D. Biochim. Biophys. Acta. 1997; 1349: 81-95Crossref PubMed Scopus (42) Google Scholar, 11Ohguchi K. Kasai T. Nozawa Y. Biochim. Biophys. Acta. 1997; 1346: 301-304Crossref PubMed Scopus (20) Google Scholar) could be due to the interaction of PA with any of these signaling molecules or perhaps a protein Tyr kinase. In this report, we examined the stimulatory role of PA on Tyr phosphorylating activity in neutrophils, characterized this activity, and identified at least one protein Tyr kinase that appears to be regulated by PA. Our findings suggest a novel facet of protein Tyr kinase regulation and a new signaling role for PA.DISCUSSIONOur understanding of the cellular roles of protein Tyr kinases is rapidly expanding because of molecular techniques that allow for the manipulation of protein expression. In contrast, our understanding of the regulatory events leading to the activation and inactivation of these signaling molecules in the intact cell lags behind. Our findings show that Tyr phosphorylating activity in human neutrophils can be regulated in a positive manner by a cell membrane-derived lipid. This represents a novel facet of protein Tyr kinase regulation. Moreover, it links protein Tyr kinase activation to stimulus-induced membrane lipid metabolism, both of which are early signaling events in most cell types.The pattern of Tyr-phosphorylated proteins observed with PMA stimulation of neutrophils was similar to that described previously (11Ohguchi K. Kasai T. Nozawa Y. Biochim. Biophys. Acta. 1997; 1346: 301-304Crossref PubMed Scopus (20) Google Scholar, 38Berkow R.L. Dodson R.W. Blood. 1990; 75: 2445-2452Crossref PubMed Google Scholar, 39Dusi S. Donini M. Rossi F. Biochem. J. 1994; 304: 243-250Crossref PubMed Scopus (46) Google Scholar). Moreover, certain proteins appear to be Tyr-phosphorylated in a manner sensitive to primary alcohols (Fig. 1). Our observations in primary neutrophils are in agreement with those made in HL-60 cells, a line with the capability to differentiate into neutrophil-like cells (11Ohguchi K. Kasai T. Nozawa Y. Biochim. Biophys. Acta. 1997; 1346: 301-304Crossref PubMed Scopus (20) Google Scholar). Together, these findings implicate phospholipase D and its product, PA, in the regulation of the Tyr phosphorylation response. In addition, exogenous PA has been shown to stimulate Tyr phosphorylation of neutrophil proteins by a mechanism that does not require its entry into cells (10Siddiqui R.A. English D. Biochim. Biophys. Acta. 1997; 1349: 81-95Crossref PubMed Scopus (42) Google Scholar). Thus, neutrophil function appears to be enhanced by PA, acting as either an intracellular messenger or an inflammatory mediator (59English D. Cell. Signal. 1996; 8: 341-347Crossref PubMed Scopus (178) Google Scholar).Our focus was on PA as an intracellular messenger. By using a cell-free system, we showed that PA stimulated Tyr phosphorylation (Fig. 2). However, PA metabolites, membrane phospholipids, and other signaling lipids failed to mimic this ability of PA (Table I). Importantly, phosphatidylalcohols, which are generated at the expense of PA upon incubation of cells with primary alcohols, also failed to mimic the ability of PA to stimulate Tyr phosphorylation (Fig. 3). Thus, the target(s) responsible for enhanced Tyr phosphorylation is (are) clearly able to discriminate among lipids (Table I and Figs. 3 and 4). This strengthens the link between the early signaling events of phospholipase D activation and Tyr phosphorylation.The activation of a protein Tyr kinase or the inhibition of a protein Tyr phosphatase provides the most straightforward explanation for PA-stimulated Tyr phosphorylation. The latter has not been reported, but PA activation of a protein Tyr phosphatase (SHP-1) has been observed (19Zhao Z. Shen S.-H. Fischer E.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4251-4255Crossref PubMed Scopus (108) Google Scholar). Since protein Tyr kinases can be activated by Tyr dephosphorylation (60Schwartzberg P.L. Oncogene. 1998; 17: 1463-1468Crossref PubMed Scopus (132) Google Scholar), this could explain our observations. However, reported properties of PA-stimulated SHP-1 activity differ significantly from those observed for PA-stimulated Tyr phosphorylation. SHP-1 activity was stimulated by PA as well as by phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and arachidonic acid and was inhibited by Mn2+ (19Zhao Z. Shen S.-H. Fischer E.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4251-4255Crossref PubMed Scopus (108) Google Scholar). In contrast, these phospholipids were poor activators of Tyr phosphorylation (Table I); arachidonic acid was inactive (Table I), and Mn2+ easily replaced Mg2+ in our assay conditions. Thus, it is unlikely that SHP-1 activation by PA is responsible for the observed lipid-stimulated increase in Tyr phosphorylation. In addition, PA-stimulated Tyr phosphorylation required the presence of ATP (not shown); a protein Tyr phosphatase inhibitor (Na3VO4) was routinely included in the Tyr phosphorylation assay mixture; and protein Tyr kinase inhibitors diminished PA-stimulated Tyr phosphorylation (Table II). These observations argue against the involvement of a protein Tyr phosphatase in the PA response.Neutrophils express numerous protein Tyr kinases (2Berton G. Curr. Opin. Hematol. 1999; 6: 51-58Crossref PubMed Scopus (27) Google Scholar, 3Welch H. Mauran C. Maridonneau-Parini I. Methods Companion Methods Enzymol. 1996; 9: 607-618Crossref Scopus (25) Google Scholar, 4Brumell J.H. Burkhardt A.L. Bolen J.B. Grinstein S. J. Biol. Chem. 1996; 271: 1455-1461Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). There is limited information on the interaction of nonreceptor protein Tyr kinases with lipids. However, some of these enzymes localize to membranes via a myristoylated amino terminus (60Schwartzberg P.L. Oncogene. 1998; 17: 1463-1468Crossref PubMed Scopus (132) Google Scholar), and Lyn and Fyn, both Src family protein Tyr kinases, have been reported to interact with glycolipids (61Kasahara K. Watanabe Y. Yamamoto T. Sanai Y. J. Biol. Chem. 1997; 272: 29947-29953Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 62Prieschl E.E. Csonga R. Novotny V. Kikuchi G.E. Baumruker T. J. Immunol. 2000; 164: 5389-5397Crossref PubMed Scopus (12) Google Scholar, 63Harder T. Scheiffele P. Verkade P. Kai S. J. Cell Biol. 1998; 141: 929-942Crossref PubMed Scopus (1040) Google Scholar). The activity of purified Src was previously reported to be enhanced by acidic phospholipids (64Sato K.-I. Yamamato H. Otsuki T. Aoto M. Tokmakov A.A. Hayashi F. Fukami Y. FEBS Lett. 1997; 410: 136-140Crossref PubMed Scopus (21) Google Scholar). Indeed, our observation that PA-stimulated Tyr phosphorylating activity is sensitive to inhibitors of protein Tyr kinases (Table II) suggests that a protein Tyr kinase is involved in the response. Among the more selective inhibitors tested, PP1, a Src family protein Tyr kinase inhibitor (48Hanke 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 (1778) Google Scholar), blocked the PA-stimulated response by nearly 50%. This suggests that a member(s) of this protein Tyr kinase family may participate in the PA-stimulated response. We observed that Fgr, a Src family enzyme, eluted exclusively in the peak of PA-dependent Tyr phosphorylating activity obtained from the gel filtration fractionation of leukocyte cytosol (Fig. 5). Moreover, PA-dependent Tyr phosphorylating activity in Fgr-containing fractions was decreased by PP1. Since no other protein Tyr kinase examined exhibited a comparable elution profile, we pursued Fgr as a candidate enzyme for regulation by PA. Fgr is primarily a membrane-associated protein (65Notario V. Gutkind J.S. Imaizumi M. Katamine S. Robbins K.C. J. Cell Biol. 1989; 109: 3129-3136Crossref PubMed Scopus (34) Google Scholar, 66Welch H. Maridonneau-Parini I. Oncogene. 1997; 15: 2021-2029Crossref PubMed Scopus (27) Google Scholar),2 yet we detected this protein Tyr kinase in the cytosolic fraction of cells disrupted by sonication. However, to maximize Fgr recovery from cells, we opted to isolate the protein by immunoprecipitation from whole cell lysates. We found that PA, but not phosphatidylbutanol, could activate Fgr (Fig.6 A). Furthermore, Fgr activation in the intact cell appears to be regulated in part by PA (Fig. 6 B). A variety of stimuli (e.g. opsonized zymosan, fMLP, PMA, the calcium ionophore A23187) are known to both elevate PA levels (12Bonser R.W. Thompson N.T. Randall R.W. Garland L.G. Biochem. J. 1989; 264: 617-620Crossref PubMed Scopus (166) Google Scholar, 40Agwu D.E. McPhail L.C. Chabot M.C. Daniel L.W. Wykle R.L. McCall C.E. J. Biol. Chem. 1989; 264: 1405-1413Abstract Full Text PDF PubMed Google Scholar, 67Regier D.S. Greene D.G. Sergeant S. Jesaitis A.J. McPhail L.C. J. Biol. Chem. 2000; 275: 28406-28412Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) and activate Fgr (66Welch H. Maridonneau-Parini I. Oncogene. 1997; 15: 2021-2029Crossref PubMed Scopus (27) Google Scholar, 68Yan S.R. Fumagalli L. Berton G. FEBS Lett. 1996; 380: 198-203Crossref PubMed Scopus (67) Google Scholar, 69Gutkind J.S. Robbins K.C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8783-8787Crossref PubMed Scopus (70) Google Scholar) in suspended neutrophils. These observations suggest that stimulus-dependent Tyr phosphorylation of proteins is mediated, in part, by PA and Fgr.The unique elution pattern of Fgr led us to discover its sensitivity to PA. However, we cannot rule out the possibility that another protein Tyr kinase(s) is regulated by PA. Several observations suggest that Fgr is not alone in its regulation by PA. These include the following: 1) PA-dependent Tyr phosphorylating activity was present in elution fractions in which Fgr was not present; 2) the selective Src family protein kinase inhibitor did not completely abolish PA-stimulated Tyr phosphorylating activity; and 3) other protein Tyr kinases coeluted with Fgr in the peak of PA-dependent Tyr phosphorylating activity.The mechanism by which PA might regulate the activity of Fgr is not yet clear. Fgr is a 55–58-kDa protein, yet it eluted from the gel filtration column in the void volume (Fig. 5). This suggests that Fgr probably associates with a protein complex. Both cellular and viral Fgr have been reported to associate with cellular proteins (70Ziemiecki A. Catelli M.-G. Joab I. Moncharmont B. Biochem. Biophys. Res. Commun. 1986; 138: 1298-1307Crossref PubMed Scopus (85) Google Scholar, 71Hartson S.D. Matts R.L. Biochemistry. 1994; 33: 8912-8920Crossref PubMed Scopus (97) Google Scholar, 72Brunati A.M. Donella-Deana A. James P. Quadroni M. Contri A. Marin O. Pinna L.A. J. Biol. Chem. 1999; 274: 7557-7564Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 73Hamada F. Aoki M. Akiyama T. Toyoshima K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6305-6309Crossref PubMed Scopus (126) Google Scholar). This raises the possibility of an indirect action of PA on the activation of Fgr, i.e. a known PA effector may be activated by PA, which in turn activates Fgr. The sensitivity of the PA-stimulated Tyr response to inhibitors of protein Ser/Thr kinases (Table II) suggests this possibility as well. Based on the data presented, we can rule out some of the known PA effectors that could potentially regulate Tyr phosphorylating activity. It is unlikely that protein kinase C is an upstream effector of a protein Tyr kinase in the cell-free system. The selective inhibitor of this protein Ser/Thr kinase had little effect on PA-stimulated Tyr phosphorylation (Table II), and the activators of protein kinase C (calcium, diacylglycerol ± PA, phosphatidylserine) were inhibitory or had no effect on PA-stimulated Tyr phosphorylation in the cell-free system. However, in the intact neutrophil, protein kinase C may play a role in the alcohol-sensitive Tyr phosphorylation due to its participation in the regulation of phospholipase D (74Frohman M.A. Sung T.-C. Morris A.J. Biochim. Biophys. Acta. 1999; 1439: 175-186Crossref PubMed Scopus (273) Google Scholar, 75Houle M.G. Bourgoin S. Biochim. Biophys. Acta. 1999; 1439: 135-150Crossref PubMed Scopus (85) Google Scholar).Among the other PA effectors, our data clearly indicate that the novel PA-activated protein Ser/Thr kinase present in neutrophil cytosol (16Waite K.A. Wallin R. Qualliotine-Mann D. McPhail L.C. J. Biol. Chem. 1997; 272: 15569-15578Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) is not an upstream regulator of a protein Tyr kinase. Activity associated with this protein kinase eluted from the gel filtration column in fractions distinct from those exhibiting PA-stimulated Tyr phosphorylation activity (Fig. 5). Finally, PKN, which is activated by free fatty acids, can also be activated by PA (76Khan W.A. Blobe G.C. Richards A.L. Hannun Y.A. J. Biol. Chem. 1994; 269: 9729-9735Abstract Full Text PDF PubMed Google Scholar). In our cell-free assay, neither arachidonic acid nor oleic acid, both potent PKN activators, had stimulatory effects on the Tyr phosphorylation (TableII) of exogenous and endogenous substrates. Thus, it is unlikely that PKN is involved in PA-stimulated Tyr phosphorylation. The data presented do not allow us to rule out an involvement of two other candidate PA effectors, Raf-1 and Pak (p21-activated kinase), which are expressed in neutrophils (16Waite K.A. Wallin R. Qualliotine-Mann D. McPhail L.C. J. Biol. Chem. 1997; 272: 15569-15578Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Experiments are underway to determine the mechanism by which PA activates Fgr.Fgr and a related Src family kinase, Hck, have been implicated in integrin signaling and cell adhesion (reviewed in Ref. 2Berton G. Curr. Opin. Hematol. 1999; 6: 51-58Crossref PubMed Scopus (27) Google Scholar). The development of a double knock-out mouse, deficient in both Fgr and Hck, has been useful in demonstrating the requirement of these protein Tyr kinases in adhesion-dependent functional responses (77Lowell C.A. Fumagalli L. Berton G. J. Cell Biol. 1996; 133: 895-910Crossref PubMed Scopus (322) Google Scholar,78Mocsai A. Ligeti E. Lowell C.A. Berton G. J. Immunol. 1999; 162: 1120-1126PubMed Google Scholar). Interestingly, ethanol, at concentrations comparable to those used in this study, has been reported to decrease neutrophil adhesion in both in situ and in vitro systems (79Brayton R.G. Stokes P.E. Schwartz M.S. Louria D.B. N. Engl. J. Med. 1970; 282: 123-128Crossref PubMed Scopus (259) Google Scholar, 80MacGregor R.R. Spagnuolo P.J. Lentnek A.L. N. Engl. J. Med. 1974; 291: 642-646Crossref PubMed Scopus (345) Google Scholar). In addition, ethanol has been shown to interfere with both oxidative and nonoxidative killing mechanisms in stimulated neutrophils (81Patel M. Keshavarzian A. Kottapalli V. Badie B. Winship D. Fields J.Z. Alcohol. Clin. Exp. Res. 1996; 20: 275-283Crossref PubMed Scopus (57) Google Scholar, 82Tamura D.Y. Moore E.E. Partrick D.A. Johnson J.L. Offner P.J. Harbeck R.J. Silliman C.C. J. Trauma Injury Infection Crit. Care. 1998; 44: 320-324Crossref PubMed Scopus (33) Google Scholar). It is tempting to speculate that an interference with PA-dependent regulation of Fgr may compromise neutrophil adhesion and adhesion-dependent cellular functions.In summary, our data indicate that human neutrophils contain Tyr phosphorylating activity that is activated by PA in the intact cell as well as in a cell-free system. Furthermore, we have identified Fgr as at least one of the protein Tyr kinases expressed in neutrophils that can be activated by PA. The lipid regulation of protein Tyr kinases (whether by a direct or indirect mechanism) constitutes a novel regulatory mechanism for this functionally important class of signaling components.AddendumWe have now observed that the magnitude of PA-stimulated Fgr activity is more variable than that shown (Fig.6 A). The variability appears to result from as yet undefined differences in lots of tissue-derived phosphatidylcholine. After testing three lots of phosphatidylcholine, we find that Fgr activity in the presence of PA is 1.45 ± 0.16 (mean ± S.E.,n = 8, p = 0.015)-fold higher than that in the presence of phosphatidylcholine. Neutrophils are dynamic, short lived cells that play a vital role in the early phases of the immune response. They provide formidable killing mechanisms against invading organisms. Neutrophils sense their environment through a variety of cell surface receptors that allow the cells to respond to bacterial products, inflammatory cytokines, immunoglobulins, complement cleavage products, and adhesion molecules. Upon receptor ligation, numerous signaling events ensue within the cell that culminate in a repertoire of functional responses appropriate for a given stimulus. Among the many signaling events induced, increased Tyr phosphorylation of proteins is commonly observed for a wide variety of neutrophil stimuli (1Rollet E. Caon A.C. Roberge C.J. Liao N.W. Malawista S.E. McColl S.R. Naccache P.H. J. Immunol. 1994; 153: 353-363PubMed Google Scholar, 2Berton G. Curr. Opin. Hematol. 1999; 6: 51-58Crossref PubMed Scopus (27) Google Scholar). Importantly, Tyr phosphorylation appears to play a central role in signaling pathways because treatment of neutrophils with inhibitors of protein Tyr kinases has been shown to interfere with most cellular functions (reviewed in Ref. 3Welch H. Mauran C. Maridonneau-Parini I. Methods Companion Methods Enzymol. 1996; 9: 607-618Crossref Scopus (25) Google Scholar). Neutrophils express a large number of nonreceptor protein Tyr kinases (2Berton G. Curr. Opin. Hematol. 1999; 6: 51-58Crossref PubMed Scopus (27) Google Scholar, 3Welch H. Mauran C. Maridonneau-Parini I. Methods Companion Methods Enzymol. 1996; 9: 607-618Crossref Scopus (25) Google Scholar, 4Brumell J.H. Burkhardt A.L. Bolen J.B. Grinstein S. J. Biol. Chem. 1996; 271: 1455-1461Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) with representatives from eight of the nine known families (5Hardie, G., and Hanks, S., eds. (1995) The Protein Kinase Facts Book, pp. 41-42, Academic Press, LondonGoogle Scholar). The functional roles of only a few of these enzymes in neutrophils have been clearly defined (reviewed in Ref. 2Berton G. Curr. Opin. Hematol. 1999; 6: 51-58Crossref PubMed Scopus (27) Google Scholar). Similarly, little is known about the regulation of protein Tyr kinases in neutrophils. The factors that influence Tyr phosphorylation in neutrophils are diverse and may reflect the variety of enzymes expressed in these cells. Examples of signaling events shown to alter protein Tyr kinase activity include ligand-induced dimerization of the Fc receptor (2Berton G. Curr. Opin. Hematol" @default.
• W1993935178 created "2016-06-24" @default.
• W1993935178 creator A5016536314 @default.
• W1993935178 creator A5033250705 @default.
• W1993935178 creator A5036396804 @default.
• W1993935178 creator A5067670293 @default.
• W1993935178 date "2001-02-01" @default.
• W1993935178 modified "2023-10-14" @default.
• W1993935178 title "Phosphatidic Acid Regulates Tyrosine Phosphorylating Activity in Human Neutrophils" @default.
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