Differential Phospholipase D Activation by Bradykinin and Sphingosine 1-Phosphate in NIH 3T3 Fibroblasts Overexpressing Gelsolin

Gelsolin, an actin-binding protein, shows a strong ability to bind to phosphatidylinositol 4,5-bisphosphate (PIP2). Here we showed in in vitroexperiments that gelsolin inhibited recombinant phospholipase D1 (PLD1) and PLD2 activities but not the oleate-dependent PLD and that this inhibition was not reversed by increasing PIP2concentration. To investigate the role of gelsolin in agonist-mediated PLD activation, we used NIH 3T3 fibroblasts stably transfected with the cDNA for human cytosolic gelsolin. Gelsolin overexpression suppressed bradykinin-induced activation of phospholipase C (PLC) and PLD. On the other hand, sphingosine 1-phosphate (S1P)-induced PLD activation could not be modified by gelsolin overexpression, whereas PLC activation was suppressed. PLD activation by phorbol myristate acetate or Ca2+ ionophore A23187 was not affected by gelsolin overexpression. Stimulation of control cells with either bradykinin or S1P caused translocation of protein kinase C (PKC) to the membranes. Translocation of PKC-α and PKC-β1 but not PKC-ε was reduced in gelsolin-overexpressed cells, whereas phosphorylation of mitogen-activated protein kinase was not changed. S1P-induced PLC activation and mitogen-activated protein kinase phosphorylation were sensitive to pertussis toxin, but PLD response was insensitive to such treatment, suggesting that S1P induced PLD activation via certain G protein distinct from Gi for PLC and mitogen-activated protein kinase pathway. Our results suggest that gelsolin modulates bradykinin-mediated PLD activation via suppression of PLC and PKC activities but did not affect S1P-mediated PLD activation. Gelsolin, an actin-binding protein, shows a strong ability to bind to phosphatidylinositol 4,5-bisphosphate (PIP2). Here we showed in in vitroexperiments that gelsolin inhibited recombinant phospholipase D1 (PLD1) and PLD2 activities but not the oleate-dependent PLD and that this inhibition was not reversed by increasing PIP2concentration. To investigate the role of gelsolin in agonist-mediated PLD activation, we used NIH 3T3 fibroblasts stably transfected with the cDNA for human cytosolic gelsolin. Gelsolin overexpression suppressed bradykinin-induced activation of phospholipase C (PLC) and PLD. On the other hand, sphingosine 1-phosphate (S1P)-induced PLD activation could not be modified by gelsolin overexpression, whereas PLC activation was suppressed. PLD activation by phorbol myristate acetate or Ca2+ ionophore A23187 was not affected by gelsolin overexpression. Stimulation of control cells with either bradykinin or S1P caused translocation of protein kinase C (PKC) to the membranes. Translocation of PKC-α and PKC-β1 but not PKC-ε was reduced in gelsolin-overexpressed cells, whereas phosphorylation of mitogen-activated protein kinase was not changed. S1P-induced PLC activation and mitogen-activated protein kinase phosphorylation were sensitive to pertussis toxin, but PLD response was insensitive to such treatment, suggesting that S1P induced PLD activation via certain G protein distinct from Gi for PLC and mitogen-activated protein kinase pathway. Our results suggest that gelsolin modulates bradykinin-mediated PLD activation via suppression of PLC and PKC activities but did not affect S1P-mediated PLD activation. phosphatidylcholine ADP-ribosylation factor Dulbecco's modified Eagle's medium gelsolin-overexposing clones 2 and 6, respectively guanosine 5′-3-O-(thio)triphosphate inositol phosphate mitogen-activated protein kinase phosphatidic acid phosphatidylbutanol phosphatidylethanolamine phosphatidylinositol 4,5-bisphosphate protein kinase C phopholipase C and D, respectively phorbol 12-myristate 13-acetate pertussis toxin sphingosine 1-phosphate vector-transfected antibody mitogen-activated protein bovine serum albumin Hydrolysis of phosphatidylcholine (PC)1 by phospholipase D (PLD) to generate choline and phosphatidic acid (PA) has been implicated in a variety of cellular functions, including secretion, vesicle trafficking, mitosis, and meiosis (1Exton J.H. J. Biol. Chem. 1997; 272: 15579-15582Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 2Exton J.H. Biochim. Biophys. Acta. 1998; 1436: 105-115Crossref PubMed Scopus (131) Google Scholar). PA has also been reported to cause activation of the growth factor signal transduction, which includes protein-tyrosine phosphatase (3Zhao Z. Shen S.H. Fischer E.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4251-4255Crossref PubMed Scopus (109) Google Scholar), phospholipase Cγ (PLCγ) (4Jones G.A. Carpenter G. J. Biol. Chem. 1993; 268: 20845-20850Abstract Full Text PDF PubMed Google Scholar), Ras-GTPase-activating protein (5Tsai M.H., Yu, C.L. Wei F.S. Stacy D.W. Science. 1989; 243: 522-526Crossref PubMed Scopus (231) Google Scholar), Raf-1 translocation (6Rizzo M.A. Shome K. Vasudevan C. Stolz D.B. Sung T.-C. Frohman M.A. Watkins S.C. Romero G. J. Biol. Chem. 1999; 274: 1131-1139Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar), and sphingosine kinase (7Melendez A. Floto R.A. Gillooly D.J. Harnett M.M. Allen J.M. J. Biol. Chem. 1998; 273: 9393-9402Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Furthermore, several lines of evidence suggest that PA induces actin polymerization based on its ability to bind to actin-binding proteins (8Cross M.J. Roberts S. Ridley A.J. Hodgkin M.N. Stewart A. Claesson-Welsh L. Wakeleam M.J.O. Curr. Biol. 1996; 6: 588-597Abstract Full Text Full Text PDF PubMed Google Scholar, 9Lee S.B. Rhee S.G. Curr. Opin. Cell Biol. 1995; 7: 183-189Crossref PubMed Scopus (284) Google Scholar, 10Barkalow K. Witke W. Kwiatkowski D.J. Hartwig J.H. J. Cell Biol. 1996; 134: 389-399Crossref PubMed Scopus (131) Google Scholar). Two mammalian PLD genes, PLD1 (11Hammond S.M. Altshuller Y.M. Sung T.C. Rudge S.A. Rose K. Engebrecht J. Morris A.J. Frohman M.A. J. Biol. Chem. 1995; 270: 29640-29643Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar) and PLD2 (12Colley W.C. Sung T.C. Roll R. Jenco J. Hammond S.M. Altshuller Y. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar, 13Kodaki T. Yamashita S. J. Biol. Chem. 1997; 272: 11408-11413Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), have recently been cloned. PLD1 is regulated by low molecular weight GTP-binding proteins such as ADP-ribosylation factor (ARF) and the Rho family and also by protein kinase C (1Exton J.H. J. Biol. Chem. 1997; 272: 15579-15582Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 2Exton J.H. Biochim. Biophys. Acta. 1998; 1436: 105-115Crossref PubMed Scopus (131) Google Scholar). The mechanisms that regulate PLD2 activity are still undefined. PLD2 is constitutively active and requires merely phosphatidylinositol 4,5-bisphosphate (PIP2) as cofactor (12Colley W.C. Sung T.C. Roll R. Jenco J. Hammond S.M. Altshuller Y. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar, 13Kodaki T. Yamashita S. J. Biol. Chem. 1997; 272: 11408-11413Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Gelsolin is a Ca2+- and polyphosphoinositide-regulated actin-binding protein (14Yin H.L. Zaner K.S. Stossel T.P. Nature. 1980; 218: 583-586Google Scholar, 15Janmey P.A. Stossel T.P. J. Biol. Chem. 1989; 264: 4825-4831Abstract Full Text PDF PubMed Google Scholar, 16Janmey P.A. Lamb J. Allen P.G. Matsudaira P.T. J. Biol. Chem. 1992; 267: 11818-11823Abstract Full Text PDF PubMed Google Scholar). In vitro studies have shown that gelsolin modulates the activities of several important signaling enzymes including PLC (17Banno Y. Nakashima T. Kumada T. Ebisawa K. Nonomura Y. Nozawa Y. J. Biol. Chem. 1992; 267: 6488-6494Abstract Full Text PDF PubMed Google Scholar, 18Sun H. Lin K. Yin H.L. J. Cell Biol. 1997; 138: 811-820Crossref PubMed Scopus (85) Google Scholar) and PLD (19Steed P.M. Nagar S. Wennogle L.P. Biochemistry. 1996; 35: 5229-5237Crossref PubMed Scopus (62) Google Scholar) through interaction with PIP2. We have previously demonstrated that gelsolin inhibited PLCγ activity via its strong binding to PIP2 in vitro (17Banno Y. Nakashima T. Kumada T. Ebisawa K. Nonomura Y. Nozawa Y. J. Biol. Chem. 1992; 267: 6488-6494Abstract Full Text PDF PubMed Google Scholar). Moreover, it has been reported that agonist-stimulated PLCβ activity was also inhibited in gelsolin-overexpressed cells (18Sun H. Lin K. Yin H.L. J. Cell Biol. 1997; 138: 811-820Crossref PubMed Scopus (85) Google Scholar). On the other hand, gelsolin has been known to enhance PLD activity through physical association (19Steed P.M. Nagar S. Wennogle L.P. Biochemistry. 1996; 35: 5229-5237Crossref PubMed Scopus (62) Google Scholar). Hydrolysis of exogenous substrate PC by PLD1 and PLD2, but not by oleate-dependent enzyme, requires the presence of PIP2 (11Hammond S.M. Altshuller Y.M. Sung T.C. Rudge S.A. Rose K. Engebrecht J. Morris A.J. Frohman M.A. J. Biol. Chem. 1995; 270: 29640-29643Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar, 12Colley W.C. Sung T.C. Roll R. Jenco J. Hammond S.M. Altshuller Y. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar, 13Kodaki T. Yamashita S. J. Biol. Chem. 1997; 272: 11408-11413Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 20Mssenburg D. Han J.S. Liyanage M.P. Patton W.A. Rhee S.G. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11717-11722Google Scholar). Thus, PLC, PLD, and gelsolin are thought to compete for available PIP2 in agonist-stimulated cells. In the present study, to determine whether gelsolin regulates PLD activity via binding to PIP2 in vivo, we examined bradykinin- and S1P-induced PLD activation in NIH 3T3 cells overexpressing gelsolin. Phosphatidylethanolamine (PE), PC, PIP2, sodium oleate, geneticin (G418), A23187, and phorbol myristate acetate (PMA) were purchased from Sigma. Sphingosine 1-phosphate (S1P) was from Matreya, Inc. (Pleasant Gap, PA). [9,10-3H]Palmitic acid (54.0 Ci/mmol),myo-[3H]inositol (90 Ci/mmol), and [choline-methyl-3H]dipalmitoyl-PC (26.5 Ci/mmol) were from NEN Life Science Products. LipofectAMINE was from Life Technologies, Inc. Rabbit polyclonal antibodies were prepared to the carboxyl-terminal 15 residues of human PLD1a (Ab-224) and carboxyl-terminal 16 residues of rat PLD2 (Ab-226). Polyclonal antibodies to PKC isozymes were from Santa Cruz Biotechnology (Santa Cruz, CA), and the antibody to phosphorylated mitogen-activated protein (MAP) kinase was from New England BioLabs (Boston, MA). Anti-rabbit antibody conjugated with horseradish peroxidase and chemiluminescence kit (ECL system) were from Amersham Pharmacia Biotech. NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 4 mml-glutamate supplemented with 10% (v/v) fetal bovine serum, 100 units of penicillin/ml, and 100 μg of streptomycin/ml at 37 °C in a humidified, CO2-controlled (5%) incubator. A HindIII-StuI fragment of human cytoplasmic gelsolin cDNA (21Cunningham C.C. Stossel T.P. Kwiatkowski D.J. Science. 1991; 251: 1233-1236Crossref PubMed Scopus (259) Google Scholar) was cloned intoHindIII-HpaI site of pLNCX retroviral vector (a generous gift from Dr. A. D. Miller, Fred Hutchinson Cancer Research Center) (22Miller A.D. Rosman G.J. Biotechniques. 1989; 7: 980-986PubMed Google Scholar). The resulting construct, or pLNCX was transfected a D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar), Two the was and to cells and NIH 3T3 cells were with the The vector and cells were in the presence of The of gelsolin or PLD proteins was examined by with antibodies the PLD1 and PLD2 were in cells that been with recombinant human PLD1 or PLD2 by Dr. M. A. of New as previously (11Hammond S.M. Altshuller Y.M. Sung T.C. Rudge S.A. Rose K. Engebrecht J. Morris A.J. Frohman M.A. J. Biol. Chem. 1995; 270: 29640-29643Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar). were in and by The were at for and the resulting was at for to the PLD1 activity was as previously D. Han J.S. Liyanage M.P. Patton W.A. Rhee S.G. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11717-11722Google Scholar) by the of choline from of PIP2, and PC in a of with [choline-methyl-3H]dipalmitoyl-PC 4 were to 100 of a PLD and 2 PLD2 activity was the used for PLD1 for and PLD activity was by the choline from [choline-methyl-3H]dipalmitoyl-PC in the presence of D. Han J.S. Liyanage M.P. Patton W.A. Rhee S.G. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11717-11722Google Scholar). NIH 3T3 cells in were with for in medium bovine serum albumin were with the and and for 15 at 37 °C with were with for the and the were by the of 10% were as by M.J. J. PubMed Scopus Google Scholar). cells were with acid in were and in (v/v) for of cells with the were by the by of of an to the were by the of and J. PubMed Scopus Google Scholar) and on a by in a an of as previously Y. K. H. A. S. Nozawa Y. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). The to by with was the and was in a were in to and to for cells were with and in sodium sodium and sodium and by were with protein as the were to on 10% and to membranes. The were with of kinase was by with a polyclonal antibody that kinase kinase was by with an antibody in and the were with horseradish the antibody was by the of PKC NIH 3T3 cells were to for The cells were and in A and and by Cell were at for and the were at for The were in A. were on by and to membranes. in the were with antibodies for PKC The were with horseradish the antibody was by the gelsolin is a actin-binding protein that has for PIP2 P.A. Stossel T.P. J. Biol. Chem. 1989; 264: 4825-4831Abstract Full Text PDF PubMed Google Scholar, 16Janmey P.A. Lamb J. Allen P.G. Matsudaira P.T. J. Biol. Chem. 1992; 267: 11818-11823Abstract Full Text PDF PubMed Google Scholar), we examined whether gelsolin PLD activity by binding the cofactor cells overexpressing PLD1 and Gelsolin inhibited PLD1 and PLD2 activities of from transfected cells as substrate On the other hand, gelsolin on oleate-dependent PLD activity in cells. showed that PLD2 activity was inhibited by gelsolin in a with inhibition at On the other hand, PLD2 activity was by PIP2 to in a with a at in the of gelsolin 2 The of gelsolin was not by increasing PIP2 suggesting that gelsolin did not PLD2 activity by binding PLD2 inhibition by gelsolin and of 2 on the PLD2 activity was with of gelsolin as substrate as the of PIP2 were examined or with gelsolin at of PIP2. are as of from the of gelsolin on PLD activation in vivo, we clones stably overexpressing gelsolin in NIH 3T3 were transfected with a gelsolin and clones and were The of gelsolin was in as by A of vector-transfected NIH 3T3 cells was used as were in the growth and and cells not To the of PLD in the transfected cells, the were to by PLD antibodies and antibodies could with recombinant human and PLD2 used as and PLD2, and and PLD2 but not PLD1a in and antibodies the presence of a of PLD2 and PLD1a of but not in vector-transfected control NIH 3T3 cells were in the of PLD and of other signaling such as PKC isozymes and and PLC isozymes and were also not and clones not To the of gelsolin overexpression on PLD activation, of was examined in and cells by The of by bradykinin and cells is shown in Gelsolin overexpression reduced induced by bradykinin with the of gelsolin showed the with control cells. PLD activation was reduced to a in which a gelsolin results suggest that inhibition of to with the of gelsolin. PLD activation is known to be on by PLC, PKC activation by from PIP2 is in the activation of PLD by S.H. P.G. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, M.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). We have previously demonstrated that gelsolin inhibited PLC activity in vitro (17Banno Y. Nakashima T. Kumada T. Ebisawa K. Nonomura Y. Nozawa Y. J. Biol. Chem. 1992; 267: 6488-6494Abstract Full Text PDF PubMed Google Scholar), and (18Sun H. Lin K. Yin H.L. J. Cell Biol. 1997; 138: 811-820Crossref PubMed Scopus (85) Google Scholar) also that gelsolin overexpression PLCβ To the of gelsolin overexpression on PLC activation in NIH 3T3 cells, we the of inositol in response to shown in bradykinin in a in control cells whereas gelsolin overexpression reduced PLC response to bradykinin by in cells of was in cells with bradykinin at To whether inhibition of PLD activation by gelsolin overexpression is a in the we examined the of Ca2+ A23187, and on PLD activation. was by with A23187 or 100 in a induced by either was not affected by gelsolin overexpression. that bradykinin-mediated PLD activation was PLC activation in NIH 3T3 S1P is known to PLD activity in cells H. A. S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, A. S. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, A. D.W. L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). To the of gelsolin on S1P signaling in NIH 3T3 cells, we PLC and PLD activation by S1P in cells. was by S1P in a in control cells but was reduced in cells at the examined by S1P in a in control cells, a with S1P at 2 In to was not inhibited in cells results that gelsolin overexpression PLC activation but not PLD activation in cells. The bradykinin has been known to to Z. K.S. J. 1997; PubMed Scopus Google Scholar), whereas the S1P with a G protein S. R. S. T. Science. 1998; PubMed Scopus Google Scholar, J. 1998; PubMed Scopus Google Scholar). A of are to G proteins such as pertussis toxin or or S. H. J. 1998; Google Scholar). shown in for of and cells reduced induced by S1P but not by suggesting that PLC activation by S1P be via G On the other hand, induced by S1P was not affected by with at a of results suggest that the G protein in PLD activation is distinct from that in PLC activation. PKC is of the of PLD activation (1Exton J.H. J. Biol. Chem. 1997; 272: 15579-15582Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). To the of PKC in the PLD activation, from bradykinin or and cells were examined by antibodies to PKC of cells with bradykinin or S1P induced translocation of and PKC-ε The of inhibited the translocation of PKC-α and PKC-β1 but not Furthermore, but not bradykinin-induced translocation of PKC isozymes was suppressed by PKC-α and PKC-β1 did not in from cells with bradykinin or On the other hand, translocation of PKC-ε induced by was not affected in gelsolin overexpressing cells. of cells with bradykinin or S1P in activation of the kinase pathway. (6Rizzo M.A. Shome K. Vasudevan C. Stolz D.B. Sung T.-C. Frohman M.A. Watkins S.C. Romero G. J. Biol. Chem. 1999; 274: 1131-1139Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar) reported that PA via activation of PLD by induced of the kinase via Raf-1 kinase activation. We examined the of gelsolin overexpression on the kinase activation an antibody that the phosphorylated of Stimulation of cells with S1P caused a phosphorylation of kinase PKC by with for and PKC but not kinase reduced S1P-induced kinase phosphorylation by of cells with S1P-induced kinase but results suggest that S1P-induced kinase activation was and shown in kinase phosphorylation induced by and S1P was in and cells, suggesting that gelsolin overexpression on agonist-stimulated kinase activation. We showed in the present that gelsolin inhibited recombinant PLD1 and PLD2 activities but not oleate-dependent PLD, which requires PIP2 as a cofactor in the exogenous substrate in vitro (1Exton J.H. J. Biol. Chem. 1997; 272: 15579-15582Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 2Exton J.H. Biochim. Biophys. Acta. 1998; 1436: 105-115Crossref PubMed Scopus (131) Google Scholar). are a of proteins for PLD activity such as and are not of PLD activity but are to reduced PIP2 in agonist-stimulated cells C. Han Morris A.J. Rhee S.G. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. Biophys. 1998; PubMed Scopus Google Scholar). also modulates agonist-mediated PLD activation by or translocation M.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, A. C. L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Nakashima S. K. Y. H. Nozawa Y. J. 1996; Google Scholar). Our study, that inhibited and PLD activities in from cells the exogenous substrate Y. Y. S. Y. Y. Nozawa Y. J. 1998; Full Text Full Text PDF PubMed Google Scholar). of was to with PIP2, the of on PLD activity could be by increasing PIP2 A. C. L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). Gelsolin is also known as a protein and PLC activity by with PIP2 (17Banno Y. Nakashima T. Kumada T. Ebisawa K. Nonomura Y. Nozawa Y. J. Biol. Chem. 1992; 267: 6488-6494Abstract Full Text PDF PubMed Google Scholar, 18Sun H. Lin K. Yin H.L. J. Cell Biol. 1997; 138: 811-820Crossref PubMed Scopus (85) Google Scholar). this is for PLD in a in PLD2 activity could not be by increasing PIP2concentration. (18Sun H. Lin K. Yin H.L. J. Cell Biol. 1997; 138: 811-820Crossref PubMed Scopus (85) Google Scholar) recently reported that PLD is with gelsolin based on PLD from we could not association with gelsolin PLD1 or PLD2 from NIH 3T3 was Our demonstrated that overexpression of gelsolin in NIH 3T3 cells modified agonist-stimulated PLC and PLD activation. Thus, gelsolin overexpression bradykinin-mediated PLC and PLD activation but did not affect PLD activation by ionophore A23187, and lines of evidence that PKC a role in PLD activation in a variety of (1Exton J.H. J. Biol. Chem. 1997; 272: 15579-15582Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 2Exton J.H. Biochim. Biophys. Acta. 1998; 1436: 105-115Crossref PubMed Scopus (131) Google Scholar). Furthermore, studies have demonstrated that PKC-α and are the of PLD activity J.H. Biochim. Biophys. Acta. 1998; 1436: 105-115Crossref PubMed Scopus (131) Google Scholar, Z. Chem. 1996; PubMed Scopus Google Scholar, K. Y. Nakashima S. Nozawa Y. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) and that the of PKC-α is an of PLD J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). PKC activation is thought to be to activation of PLC which PIP2 to generate inositol and The in and Ca2+ to activation and translocation of PKC Thus, the interaction of PKC with PLD in be to PLD activation. PLD activation was in cells, suggesting that PLC of PLD. The of in PLD by growth has been shown by studies fibroblasts with Carpenter G. J.H. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). Our studies demonstrated that gelsolin overexpression inhibited PLCβ to of activation of PKC-α and PKC-β1 but not the reduced PLD response to bradykinin in gelsolin-overexpressed cells be to PKC-α activation. was by the that or PLD activation was by gelsolin overexpression. results suggest that bradykinin-mediated PLD activation be to PLD1 activation is by several such as PKC and G proteins Rho and (1Exton J.H. J. Biol. Chem. 1997; 272: 15579-15582Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 2Exton J.H. Biochim. Biophys. Acta. 1998; 1436: 105-115Crossref PubMed Scopus (131) Google Scholar, K. Y. Nakashima S. Nozawa Y. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, S.M. Jenco J.M. Nakashima S. K. S. Nozawa Y. Frohman M.A. Morris A.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). We also that bradykinin in of control cells, whereas its was in gelsolin-overexpressed cells not reduced be also for the reduced PLD response to bradykinin in gelsolin cells. On the other hand, PLD2 is of PLC activation, it is not by PKC and G proteins (1Exton J.H. J. Biol. Chem. 1997; 272: 15579-15582Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 2Exton J.H. Biochim. Biophys. Acta. 1998; 1436: 105-115Crossref PubMed Scopus (131) Google Scholar). S1P induced PLC and PLD activation and translocation of PKC in NIH 3T3 cells. Gelsolin overexpression reduced PLC response to S1P and translocation of PKC-α and PKC-β1 but did not affect PLD activation. results that S1P-induced PLD activation was and PLD2, which was present in cells. Furthermore, suggesting that PLD2 is in S1P-induced PLD activation. In NIH 3T3 cells transfected with PLD2 was in S1P-induced PLD activation, whereas cells transfected with PLD2 showed reduced PLD activation in response to S1P not suggest that gelsolin bradykinin-induced PLD1 activation via suppression but not affect S1P-induced PLD2 activation. Our that S1P-mediated signaling activation of PLC, and kinase was sensitive to but PLD activation was insensitive to such that PLD activation is of signaling is with the S1P signaling in 3T3 fibroblasts A. S. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). are of S1P and S. H. J. 1998; Google Scholar). is to protein, which the signaling PLC, and kinase H. K. H. K. Y. H. Y. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). In and the via the and S. H. J. 1998; Google Scholar, K. H. Y. H. T. S. R. K. Y. J. 1999; PubMed Scopus Google Scholar). Furthermore, it has been reported that in cells stably S1P induces kinase activation in a and but A. S. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). In demonstrated that kinase activation was and but results that in S1P-induced kinase activation was via Gi but not PLC activation induced by bradykinin and S1P were suppressed by gelsolin overexpression, whereas kinase activation by was not results suggest that S1P induced PLD activation via certain G proteins distinct from Gi for PLC and the kinase in NIH 3T3 PLD2 was demonstrated to be in kinase (8Cross M.J. Roberts S. Ridley A.J. Hodgkin M.N. Stewart A. Claesson-Welsh L. Wakeleam M.J.O. Curr. Biol. 1996; 6: 588-597Abstract Full Text Full Text PDF PubMed Google Scholar). Our that PLC PLD is with kinase activation in NIH 3T3 We Dr. M. A. Frohman of New for cDNA of human PLD1 and