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• W2018149384 abstract "Stimulation of phospholipase D (PLD) by membrane receptors is now recognized as a major signal transduction pathway involved in diverse cellular functions. Rho proteins control receptor signaling to PLD, and these GTPases have been shown to directly stimulate purified recombinant PLD1 enzymes in vitro. Here we report that stimulation of PLD activity, measured in the presence of phosphatidylinositol 4,5-bisphosphate, by RhoA in membranes of HEK-293 cells expressing the m3 muscarinic acetylcholine receptor (mAChR) is phosphorylation-dependent. Therefore, the possible involvement of the RhoA-stimulated serine/threonine kinase, Rho-kinase, was investigated. Overexpression of Rho-kinase and constitutively active Rho-kinase (Rho-kinase-CAT) but not of kinase-deficient Rho-kinase-CAT markedly increased m3 mAChR-mediated but not protein kinase C-mediated PLD stimulation, similar to overexpression of RhoA. Expression of the Rho-inactivating C3 transferase abrogated the stimulatory effect of wild-type Rho-kinase, but not of Rho-kinase-CAT. Recombinant Rho-kinase-CAT mimicked the phosphorylation-dependent PLD stimulation by RhoA in HEK-293 cell membranes. Finally, the Rho-kinase inhibitor HA-1077 largely inhibited RhoA-induced PLD stimulation in membranes as well as PLD stimulation by the m3 mAChR but not by protein kinase C in intact HEK-293 cells. We conclude that Rho-kinase is involved in Rho-dependent PLD stimulation by the G protein-coupled m3 mAChR in HEK-293 cells. Thus, our findings identify Rho-kinase as a novel player in the receptor-controlled PLD signaling pathway. Stimulation of phospholipase D (PLD) by membrane receptors is now recognized as a major signal transduction pathway involved in diverse cellular functions. Rho proteins control receptor signaling to PLD, and these GTPases have been shown to directly stimulate purified recombinant PLD1 enzymes in vitro. Here we report that stimulation of PLD activity, measured in the presence of phosphatidylinositol 4,5-bisphosphate, by RhoA in membranes of HEK-293 cells expressing the m3 muscarinic acetylcholine receptor (mAChR) is phosphorylation-dependent. Therefore, the possible involvement of the RhoA-stimulated serine/threonine kinase, Rho-kinase, was investigated. Overexpression of Rho-kinase and constitutively active Rho-kinase (Rho-kinase-CAT) but not of kinase-deficient Rho-kinase-CAT markedly increased m3 mAChR-mediated but not protein kinase C-mediated PLD stimulation, similar to overexpression of RhoA. Expression of the Rho-inactivating C3 transferase abrogated the stimulatory effect of wild-type Rho-kinase, but not of Rho-kinase-CAT. Recombinant Rho-kinase-CAT mimicked the phosphorylation-dependent PLD stimulation by RhoA in HEK-293 cell membranes. Finally, the Rho-kinase inhibitor HA-1077 largely inhibited RhoA-induced PLD stimulation in membranes as well as PLD stimulation by the m3 mAChR but not by protein kinase C in intact HEK-293 cells. We conclude that Rho-kinase is involved in Rho-dependent PLD stimulation by the G protein-coupled m3 mAChR in HEK-293 cells. Thus, our findings identify Rho-kinase as a novel player in the receptor-controlled PLD signaling pathway. Phospholipase D (PLD) 1The abbreviations used are: PLD, phospholipase D; ARF, ADP ribosylation factor; GTPγS, guanosine 5′-O-(3-thio)triphosphate; mAChR, muscarinic acetylcholine receptor; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PtdCho, phosphatidylcholine; PtdEtOH, phosphatidylethanol; PtdIns4P, phosphatidylinositol 4-phosphate; PtdIns(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; Rho-kinase-CAT, catalytic domain of Rho-kinase; Rho-kinase-CAT-KD, kinase-deficient mutant of Rho-kinase-CAT; GST, glutathioneS-transferase. enzymes belong to a newly identified enzyme family known to exist in plant, bacteria, yeast, and mammalian sources. Stimulation of PLD has been described in many cellular systems in response to a large variety of agonist-activated tyrosine kinase receptors and receptors coupled to heterotrimeric G proteins and is apparently involved in various signaling processes (1Morris A.J. Engebrecht J. Frohman M.A. Trends Pharmacol. Sci. 1996; 17: 182-185Abstract Full Text PDF PubMed Scopus (176) Google Scholar, 2Cockcroft S. Prog. Lipid Res. 1997; 35: 345-370Crossref Scopus (56) Google Scholar, 3Exton J.H. J. Biol. Chem. 1997; 272: 15579-15582Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Specifically, PLD and its immediate reaction product, phosphatidic acid, have been reported to regulate diverse cellular events, such as vesicular trafficking, actin stress fiber formation, activation of Raf-1 kinase, and phosphatidylinositol 4-phosphate (PtdIns4P) 5-kinase isoforms, to name but a few (4Moritz 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, 5Cross M.J. Roberts S. Ridley A.J. Hodgkin M.N. Stewart A. Claesson-Walsh L. Wakelam M.J.O. Curr. Biol. 1996; 6: 588-597Abstract Full Text Full Text PDF PubMed Google Scholar, 6Gosh S. Strum J.C. Sciorra V.A. Daniel L. Bell R.M. J. Biol. Chem. 1996; 271: 8472-8480Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 7Fensome A. Cunningham E. Prosser S. Tan S.K. Swigart P. Thomas G. Hsuan J. Cockcroft S. Curr. Biol. 1996; 6: 730-738Abstract Full Text Full Text PDF PubMed Google Scholar, 8Ktistakis N.T. Brown H.A. Waters M.G. Sternweis P.C. Roth M.G. J. Cell Biol. 1996; 134: 295-306Crossref PubMed Scopus (329) Google Scholar, 9Chen Y.-G. Siddhanta A. Austin C.D. Hammond S.M. Sung T.-C. Frohman M.A. Morris A.J. Shields D. J. Cell Biol. 1997; 138: 495-594Crossref PubMed Scopus (242) Google Scholar). The two mammalian PLD isoforms identified thus far, PLD1 (with the two splice variants PLD1a and PLD1b) and PLD2, differ greatly in their regulatory properties. PLD2 is thought to be solely stimulated by the phosphoinositide phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2; Refs. 10Colley 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 and 11Kodaki T. Yamashita S. J. Biol. Chem. 1997; 272: 11408-11413Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), although recent reports on cloned PLD2 enzymes suggest that this PLD isoform may also be activated, but very modestly, by ADP ribosylation factor (ARF), a member of the low molecular weight GTPase superfamily (12Lopez I. Arnold R.S. Lambeth J.D. J. Biol. Chem. 1998; 273: 12846-12852Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 13Sung T.-C. Altshuller Y.M. Morris A.J. Frohman M.A. J. Biol. Chem. 1999; 274: 494-502Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). On the other hand, PLD1 enzymes are strongly stimulated by PtdIns(4,5)P2 and ARF and, in addition, by some protein kinase C (PKC) isoforms and by Rho GTPases (14Hammond 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, 15Park S.-K. Provost J.J. Bae C.D. Ho W.-T. Exton J.H. J. Biol. Chem. 1997; 272: 29263-29271Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Specifically, the Rho family GTPases RhoA, Rac1, and Cdc42, which are activated by the stable GTP analog guanosine 5′-O-(3-thio)triphosphate (GTPγS), have been shown to stimulate purified recombinant PLD1 enzymes, apparently by a direct interaction of PLD1 with these GTPases (16Hammond S.M. Jenco J.M. Nakashima S. Cadwallader K. Gu Q. Cook S. Nozawa Y. Prestwich G.D. Frohman M.A. Morris A.J. J. Biol. Chem. 1997; 272: 3860-3868Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar, 17Sung T.-C. Roper R.L. Zhang Y. Rudge S.A. Temel R. Hammond S.M. Morris A.J. Moss B. Engebrecht J. Frohmann M.A. EMBO J. 1997; 16: 4519-4530Crossref PubMed Scopus (305) Google Scholar, 18Min D.S. Park S.-K. Exton J.H. J. Biol. Chem. 1998; 273: 7044-7051Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 19Bae C.D. Min D.S. Fleming I.N. Exton J.H. J. Biol. Chem. 1998; 273: 11596-11604Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Furthermore, studies performed with toxins inactivating Rho GTPases indicated that these GTPases are also involved in PLD stimulation by G protein-coupled and growth factor receptors in intact cells (20Schmidt M. Rümenapp U. Bienek C. Keller J. von Eichel-Streiber C. Jakobs K.H. J. Biol. Chem. 1996; 271: 2422-2426Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 21Malcolm K.C. Elliott C.M. Exton J.H. J. Biol. Chem. 1996; 271: 13135-13139Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 22Hess J.A. Ross A.H. Qui R.-G. Symons M. Exton J.H. J. Biol. Chem. 1997; 272: 1615-1620Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 23Fensome A. Whatmore J. Morgan C. Jones D. Cockcroft S. J. Biol. Chem. 1998; 273: 13157-13164Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). However, stimulation of endogenous PLD by Rho GTPases seems to be rather complex. Whereas RhoA stimulation of ARF-sensitive PLD has been reported in some cell-free systems (24Bowman E.P. Uhlinger D.J. Lambeth J.D. J. Biol. Chem. 1993; 268: 21509-21512Abstract Full Text PDF PubMed Google Scholar, 25Malcolm K.C. Ross A.H. Qui R.G. Symons M. Exton J.H. J. Biol. Chem. 1994; 269: 25951-25954Abstract Full Text PDF PubMed Google Scholar, 26Provost J.J. Fudge J. Israelit S. Siddiqi A.R. Exton J.H. Biochem. J. 1996; 319: 285-291Crossref PubMed Scopus (72) Google Scholar), it was without effect in others or could even be resolved from the ARF-stimulated PLD (15Park S.-K. Provost J.J. Bae C.D. Ho W.-T. Exton J.H. J. Biol. Chem. 1997; 272: 29263-29271Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 26Provost J.J. Fudge J. Israelit S. Siddiqi A.R. Exton J.H. Biochem. J. 1996; 319: 285-291Crossref PubMed Scopus (72) Google Scholar, 27Vinggaard A.M. Provost J.J. Exton J.H. Hansen H.S. Cell. Signal. 1997; 9: 189-196Crossref PubMed Scopus (17) Google Scholar). Rho GTPases may also indirectly stimulate PLD enzymes by increasing the synthesis of PtdIns(4,5)P2 by PtdIns4P 5-kinases (28Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Crossref PubMed Scopus (576) Google Scholar), which has been demonstrated in various cellular systems to be of crucial importance for signaling to PLD (2Cockcroft S. Prog. Lipid Res. 1997; 35: 345-370Crossref Scopus (56) Google Scholar, 3Exton J.H. J. Biol. Chem. 1997; 272: 15579-15582Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 7Fensome A. Cunningham E. Prosser S. Tan S.K. Swigart P. Thomas G. Hsuan J. Cockcroft S. Curr. Biol. 1996; 6: 730-738Abstract Full Text Full Text PDF PubMed Google Scholar, 29Pertile P. Liscovitch M. Chalifa V. Cantley L.C. J. Biol. Chem. 1995; 270: 5130-5135Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 30Schmidt M. Rümenapp U. Nehls C. Ott S. Keller J. von Eichel-Streiber C. Jakobs K.H. Eur. J. Biochem. 1996; 240: 707-712Crossref PubMed Scopus (42) Google Scholar). We have recently reported that in HEK-293 cells stably expressing the G protein-coupled m3 muscarinic acetylcholine receptor (mAChR), PLD activity depends on PtdIns(4,5)P2 and that PLD stimulation by phorbol ester-activated PKC involves the Ras-related Ral proteins, whereas m3 mAChR signaling to PLD is mediated by members of the ARF and Rho GTPase families (20Schmidt M. Rümenapp U. Bienek C. Keller J. von Eichel-Streiber C. Jakobs K.H. J. Biol. Chem. 1996; 271: 2422-2426Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 30Schmidt M. Rümenapp U. Nehls C. Ott S. Keller J. von Eichel-Streiber C. Jakobs K.H. Eur. J. Biochem. 1996; 240: 707-712Crossref PubMed Scopus (42) Google Scholar, 31Schmidt M. Hüwe S.M. Fasselt B. Homann D. Rümenapp U. Sandmann J. Jakobs K.H. Eur. J. Biochem. 1994; 225: 667-675Crossref PubMed Scopus (89) Google Scholar, 32Rümenapp U. Schmidt M. Wahn F. Tapp E. Grannass A. Jakobs K.H. Eur. J. Biochem. 1997; 248: 407-414Crossref PubMed Scopus (23) Google Scholar, 33Schmidt M. Voβ M. Thiel M. Bauer B. Grannaβ A. Tapp E. Cool R.H. de Gunzburg J. von Eichel-Streiber C. Jakobs K.H. J. Biol. Chem. 1998; 273: 7413-7422Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 34Rümenapp U. Geiszt M. Wahn F. Schmidt M. Jakobs K.H. Eur. J. Biochem. 1995; 234: 240-244Crossref PubMed Scopus (81) Google Scholar). The aim of the present study was to identify the mechanism of PLD stimulation by Rho proteins in HEK-293 cells. We demonstrate here that the PLD stimulatory effect of recombinant RhoA in HEK-293 cell membranes is phosphorylation-dependent. In the search for the putative kinase, we studied the effect of the RhoA-stimulated serine/threonine kinase Rho-kinase (35Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (943) Google Scholar), which has also been termed ROKα (36Leung T. Manser E. Tan L. Lim L. J. Biol. Chem. 1995; 270: 29051-29054Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar) and ROCK-II (37Nakagawa O. Fujisawa K. Ishizaki T. Saito Y. Nakao K. Narumiya S. FEBS Lett. 1996; 392: 189-193Crossref PubMed Scopus (657) Google Scholar), on PLD regulation. We found that overexpression of Rho-kinase greatly increases m3 mAChR-mediated but not PKC-mediated PLD stimulation in intact cells. Furthermore, we show that HA-1077, a Rho-kinase inhibitor, specifically suppresses receptor-mediated PLD stimulation and that recombinant Rho-kinase mimics the stimulatory effect of RhoA on PLD activity in HEK-293 cell membranes. These findings strongly suggest that Rho-kinase is involved in Rho-controlled PLD stimulation by the G protein-coupled m3 mAChR in HEK-293 cells. [3H]Oleic acid (10 Ci/mmol) and 1-palmitoyl-2-[3H]palmitoyl-glycerophosphocholine ([3H]PtdCho; 37.5 Ci/mmol) were from New England Nuclear. HA-1077 was from Calbiochem, and glutathione-Sepharose was from Amersham Pharmacia Biotech. Unlabeled PtdCho, phorbol 12-myristate 13-acetate (PMA), and TNM-FH insect medium were from Sigma, and PtdIns(4,5)P2 and GTPγS were from Roche Molecular Biochemicals. Antibodies against RhoA and Rho-kinase were purchased from Santa Cruz Biotechnology. DNA encoding human RhoA was subcloned into pRK5 expression vector. DNA encoding myc-tagged C3 transferase subcloned in pEF (38Hill C.S. Wynne J. Treismann R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1207) Google Scholar) was a kind gift of Dr. A. Hall. DNAs encoding myc-tagged wild-type Rho-kinase, the catalytic domain of Rho-kinase, Rho-kinase-CAT (amino acids 6–553), and the kinase-deficient mutant of Rho-kinase-CAT, Rho-kinase-CAT-KD (Rho-kinase-CAT K121G), were subcloned into pEF (39Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1685) Google Scholar). For expression in Sf9 cells, DNA encoding RhoA was subcloned into a pAcGHLT baculovirus transfer vector (PharMingen), and DNA encoding Rho-kinase-CAT was subcloned into a pAcGLT transfer vector (39Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1685) Google Scholar). Culture conditions of HEK-293 cells stably expressing the m3 mAChR were as reported previously (31Schmidt M. Hüwe S.M. Fasselt B. Homann D. Rümenapp U. Sandmann J. Jakobs K.H. Eur. J. Biochem. 1994; 225: 667-675Crossref PubMed Scopus (89) Google Scholar). For experiments, cells subcultured in Dulbecco's modified Eagle's medium/F-12 medium were grown to near confluence (145-mm culture dishes) and transfected with either the indicated concentrations of DNA encoding RhoA, myc-tagged C3 transferase, Rho-kinase, Rho-kinase-CAT or Rho-kinase-CAT-KD, or the corresponding vectors using the calcium phosphate method (40Winstel R. Freund S. Krasel C. Hoppe E. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2105-2109Crossref PubMed Scopus (145) Google Scholar). Transfection efficiency of HEK-293 cells, which ranged from 50% to 80%, was determined by in situ staining for β-galactosidase activity of the cells cotransfected with the constitutively active pSVβ-gal (Promega). All assays were performed 48 h after transfection. Transient overexpression of the proteins was verified by immunoblotting of cell lysates using specific antibodies. Transient overexpression of C3 transferase was detected by the mobility shift of ADP-ribosylated endogenously expressed RhoA (41Aktories K. Just I. Dickey B.F. Birnbaumer L. GTPases in Biology I. Springer-Verlag, Berlin1993: 87-112Google Scholar). Morphological changes induced by overexpression of RhoA and Rho-kinase were visualized by phase-contrast microscopy (Nikon TMS). For measurement of PLD activity in intact HEK-293 cells, the cells were replated 24 h after transfection on 145-mm culture dishes. Cellular phospholipids were labeled by incubating monolayers for 20–24 h with [3H]oleic acid (2 μCi/ml) in growth medium. Thereafter, cells were detached from the dishes, washed twice in Hank's balanced salt solution containing 118 mm NaCl, 5 mm KCl, 1 mm CaCl2, 1 mm MgCl2, and 5 mmd-glucose buffered at pH 7.4 with 15 mm HEPES, and resuspended at a cell concentration of 1 × 107 cells/ml. PLD activity was measured for 60 min at 37 °C in a total volume of 200 μl containing 100 μl of cell suspension (1 × 106 cells), 400 mmethanol, and the indicated stimulatory agents. The reaction was stopped, and labeled phospholipids, including the specific PLD product [3H]phosphatidylethanol ([3H]PtdEtOH), were isolated as described previously (31Schmidt M. Hüwe S.M. Fasselt B. Homann D. Rümenapp U. Sandmann J. Jakobs K.H. Eur. J. Biochem. 1994; 225: 667-675Crossref PubMed Scopus (89) Google Scholar). The formation of [3H]PtdEtOH is expressed as a percentage of the total amount of labeled phospholipids. Data shown are the mean ± S.D. from one experiment performed in triplicate and repeated as indicated in the figure legends. To measure PLD activity in HEK-293 cell membranes prepared as described previously (30Schmidt M. Rümenapp U. Nehls C. Ott S. Keller J. von Eichel-Streiber C. Jakobs K.H. Eur. J. Biochem. 1996; 240: 707-712Crossref PubMed Scopus (42) Google Scholar), [3H] PtdCho was mixed with PtdIns(4,5)P2 in a molar ratio of 8:1, dried, and resuspended in 50 mmHEPES, pH 7.5, 3 mm EGTA, 80 mm KCl, and 1 mm dithiothreitol, followed by sonication on ice. PLD activity was determined as described previously (30Schmidt M. Rümenapp U. Nehls C. Ott S. Keller J. von Eichel-Streiber C. Jakobs K.H. Eur. J. Biochem. 1996; 240: 707-712Crossref PubMed Scopus (42) Google Scholar) with [3H]PtdCho/PtdIns(4,5)P2 (200 μm/25 μm) as substrate vesicles and 200 μg of membrane protein for 60 min at 37 °C or for 15 min at 30 °C. Sf9 cells (1 × 106 cells/ml) cultured at 25 °C in TNM-FH insect medium containing 10% fetal calf serum, 100 units/ml penicillin G, and 100 μg/ml streptomycin were infected with pAcGHLT containing RhoA or pAcGLT containing Rho-kinase-CAT baculovirus transfer vectors (multiplicity of infection = 5) for 48 h at 25 °C. Thereafter, the cells were centrifuged, resuspended in Buffer A (50 mm NaCl, 10 mm MgCl2, 1 mm dithiothreitol, 10 μm phenylmethylsulfonyl fluoride, and 10 mm Tris-HCl, pH 7.5), and homogenized by sonication on ice. The lysates were centrifuged for 1 h at 20,000 × g. The supernatant, which contained GST-RhoA or GST-Rho-kinase-CAT, was incubated with glutathione-Sepharose beads for 30 min at 4 °C. Thereafter, the beads were washed three times with Buffer A to remove unbound proteins. RhoA and Rho-kinase-CAT were released from the parent GST-fusion proteins bound to the beads by incubation with thrombin (PharMingen; 10 units) overnight at 4 °C in a buffer containing 150 mm NaCl, 5 mmMgCl2, 2.5 mm CaCl2, 1 mm dithiothreitol, and 50 mm Tris-HCl, pH 8.0. The beads were removed by centrifugation, and the excess thrombin was removed by the addition of p-aminobenzamidine beads. The homogeneity of the recombinant RhoA and Rho-kinase-CAT proteins was analyzed by SDS-polyacrylamide gel electrophoresis. For immunoblot analysis, an aliquot of the homogenates was subjected to SDS-polyacrylamide gel electrophoresis on 10% acrylamide gels to separate the proteins. After a transfer to nitrocellulose membranes and a 1-h incubation with anti-RhoA (1:500 dilution) or anti-Rho-kinase (1:200 dilution) antibodies, the proteins were visualized by enhanced chemiluminescence. We have reported previously that PLD stimulation by the m3 mAChR, which activates endogenous RhoA in HEK-293 cells (42Keller J. Schmidt M. Hussein B. Rümenapp U. Jakobs K.H. FEBS Lett. 1997; 403: 299-302Crossref PubMed Scopus (32) Google Scholar), is potently inhibited by the inactivation of Rho family GTPases with Clostridium difficile toxin B (20Schmidt M. Rümenapp U. Bienek C. Keller J. von Eichel-Streiber C. Jakobs K.H. J. Biol. Chem. 1996; 271: 2422-2426Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Furthermore, it has been shown that toxin B and the Rho-specific C3 transferase (41Aktories K. Just I. Dickey B.F. Birnbaumer L. GTPases in Biology I. Springer-Verlag, Berlin1993: 87-112Google Scholar) decrease the cellular level of PtdIns(4,5)P2 and that PtdIns(4,5)P2 regulates PLD activity in HEK-293 cell membranes (30Schmidt M. Rümenapp U. Nehls C. Ott S. Keller J. von Eichel-Streiber C. Jakobs K.H. Eur. J. Biochem. 1996; 240: 707-712Crossref PubMed Scopus (42) Google Scholar, 32Rümenapp U. Schmidt M. Wahn F. Tapp E. Grannass A. Jakobs K.H. Eur. J. Biochem. 1997; 248: 407-414Crossref PubMed Scopus (23) Google Scholar, 43Schmidt M. Bienek C. Rümenapp U. Zhang C. Lümmen G. Jakobs K.H. Just I. Aktories K. Moos M. von Eichel-Streiber C. Naunyn-Schmiedeberg's Arch. Pharmacol. 1996; 354: 87-94Crossref PubMed Scopus (44) Google Scholar). Thus, to study PLD stimulation by Rho proteins in HEK-293 cells, we measured PLD activities in the membranes of HEK-293 cells in the presence of PtdIns(4,5)P2. Several previous studies have demonstrated that activated RhoA stimulates purified recombinant PLD1 enzymes under this condition, apparently by a direct RhoA-PLD1 interaction (16Hammond S.M. Jenco J.M. Nakashima S. Cadwallader K. Gu Q. Cook S. Nozawa Y. Prestwich G.D. Frohman M.A. Morris A.J. J. Biol. Chem. 1997; 272: 3860-3868Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar, 17Sung T.-C. Roper R.L. Zhang Y. Rudge S.A. Temel R. Hammond S.M. Morris A.J. Moss B. Engebrecht J. Frohmann M.A. EMBO J. 1997; 16: 4519-4530Crossref PubMed Scopus (305) Google Scholar, 18Min D.S. Park S.-K. Exton J.H. J. Biol. Chem. 1998; 273: 7044-7051Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 19Bae C.D. Min D.S. Fleming I.N. Exton J.H. J. Biol. Chem. 1998; 273: 11596-11604Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In HEK-293 cell membranes, the addition of GTPγS (100 μm) alone caused about a 2-fold increase in PLD activity, which is probably due to the activation of endogenous membrane-associated ARF proteins (30Schmidt M. Rümenapp U. Nehls C. Ott S. Keller J. von Eichel-Streiber C. Jakobs K.H. Eur. J. Biochem. 1996; 240: 707-712Crossref PubMed Scopus (42) Google Scholar, 32Rümenapp U. Schmidt M. Wahn F. Tapp E. Grannass A. Jakobs K.H. Eur. J. Biochem. 1997; 248: 407-414Crossref PubMed Scopus (23) Google Scholar). Surprisingly, however, the addition of purified recombinant RhoA (10 μm) in the presence of GTPγS (100 μm) had no effect on PLD activity (Fig. 1). Similar data were obtained in the membranes of HEK-293 cells pretreated with toxin B, causing the inactivation of endogenous Rho proteins (data not shown). In contrast, under the same assay conditions, GTPγS-activated recombinant RhoA stimulated PLD activity in the membranes of human PLD1a-expressing Sf9 cells about 20-fold, from 50 ± 10 to 1070 ± 80 pmol × h−1 × mg protein−1 (mean ± SD; n = 5 experiments). Besides PtdIns4P 5-kinases, Rho GTPases can stimulate various protein kinases (for reviews, see Refs. 28Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Crossref PubMed Scopus (576) Google Scholar and 44Lim L. Manser E. Leung T. Hall C. Eur. J. Biochem. 1996; 242: 171-185Crossref PubMed Scopus (273) Google Scholar). Therefore, we studied whether a phosphorylation reaction is involved in RhoA stimulation of HEK-293 cell PLD activity. For this study, PLD activity was measured in the presence of 1 mm MgATP. Under this condition, the addition of RhoA in the presence of GTPγS markedly increased PLD activity in HEK-293 cell membranes (Fig. 1). Although a permissive effect of MgATP on GTPγS binding by RhoA cannot be excluded, we first considered the possibility that a RhoA-dependent lipid or protein kinase is involved in PLD stimulation by RhoA. The involvement of a RhoA-stimulated PtdIns4P 5-kinase was unlikely for the following reasons: (a) PtdIns(4,5)P2 was added at a maximally effective concentration of 25 μm (30Schmidt M. Rümenapp U. Nehls C. Ott S. Keller J. von Eichel-Streiber C. Jakobs K.H. Eur. J. Biochem. 1996; 240: 707-712Crossref PubMed Scopus (42) Google Scholar); (b) although the added PtdIns(4,5)P2 was degraded at the end of the incubation period in the absence of MgATP to 10 μm, this PtdIns(4,5)P2 concentration was virtually identical to that (12 μm) used by many others to demonstrate RhoA stimulation of PLD in vitro (16Hammond S.M. Jenco J.M. Nakashima S. Cadwallader K. Gu Q. Cook S. Nozawa Y. Prestwich G.D. Frohman M.A. Morris A.J. J. Biol. Chem. 1997; 272: 3860-3868Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar, 17Sung T.-C. Roper R.L. Zhang Y. Rudge S.A. Temel R. Hammond S.M. Morris A.J. Moss B. Engebrecht J. Frohmann M.A. EMBO J. 1997; 16: 4519-4530Crossref PubMed Scopus (305) Google Scholar, 18Min D.S. Park S.-K. Exton J.H. J. Biol. Chem. 1998; 273: 7044-7051Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 19Bae C.D. Min D.S. Fleming I.N. Exton J.H. J. Biol. Chem. 1998; 273: 11596-11604Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar,45Brown H.A. Gutowski S. Moomaw C.R. Slaughter C. Sternweis P.C. Cell. 1993; 75: 1137-1144Abstract Full Text PDF PubMed Scopus (823) Google Scholar); and (c) in the presence of MgATP, which by itself prevented (by ∼50%) the degradation of added PtdIns(4,5)P2, the addition of RhoA had a very modest protective effect on the PtdIns(4,5)P2 level (data not shown), which was quite distinct from the marked PLD stimulation by RhoA under this condition. Thus, the phosphorylation-dependent PLD stimulation by RhoA in HEK-293 cell membranes may involve a protein kinase rather than PtdIns4P 5-kinase. Recently, several direct RhoA target proteins have been identified, including the two Rho-stimulated serine/threonine kinases: (a) Rho-kinase, also termed ROKα or ROCK-II, and (b) p160ROCK, also termed ROCK-I (35Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (943) Google Scholar, 36Leung T. Manser E. Tan L. Lim L. J. Biol. Chem. 1995; 270: 29051-29054Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar, 37Nakagawa O. Fujisawa K. Ishizaki T. Saito Y. Nakao K. Narumiya S. FEBS Lett. 1996; 392: 189-193Crossref PubMed Scopus (657) Google Scholar, 46Ishizaki T. Maekawa M. Fujisawa K. Okawa K. Iwamatsu A. Fujita A. Watanabe N. Saito Y. Kakizuka A. Morii N. Narumiya S. EMBO J. 1996; 15: 1885-1893Crossref PubMed Scopus (796) Google Scholar). To test the hypothesis that Rho-induced PLD stimulation involves Rho-kinase, we studied the effects of HEK-293 cell transfection with RhoA and different Rho-kinase constructs on cell morphology and PLD activity. Expression of RhoA and the myc-tagged Rho-kinases was verified by immunofluorescence and immunoblotting (data not shown). Overexpression of RhoA caused drastic changes in HEK-293 cell morphology, as demonstrated by the occurrence of a high number of rounded cells (Fig. 2). As reported previously by others (36Leung T. Manser E. Tan L. Lim L. J. Biol. Chem. 1995; 270: 29051-29054Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar,44Lim L. Manser E. Leung T. Hall C. Eur. J. Biochem. 1996; 242: 171-185Crossref PubMed Scopus (273) Google Scholar, 47Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (951) Google Scholar), similar morphology changes were observed in HEK-293 cells overexpressing Rho-kinase-CAT, which lacks the regulatory Rho-binding and PH domains and is constitutively active, and although less pronounced, in wild-type Rho-kinase-overexpressing cells. In contrast, overexpression of a kinase-deficient mutant of Rho-kinase-CAT, Rho-kinase-CAT-KD, did not cause rounding of HEK-293 cells. We then determined the effects of the same proteins on PLD activity in HEK-293 cells. As illustrated in Fig. 3, overexpression of RhoA and either wild-type Rho-kinase or Rho-kinase-CAT markedly increased m3 mAChR-mediated PLD stimulation without significantly altering basal PLD activity. The PLD stimulatory effects were most pronounced in cells overexpressing RhoA and Rho-kinase-CAT. For example, the transfection of HEK-293 cells with 100 μg of RhoA DNA and Rho-kinase-CAT DNA increased PLD stimulation by carbachol (1 mm) by about 150% and 250%, respectively (Fig. 3, A and C). A significant but less pronounced increase in m3 m" @default.
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• W2018149384 title "A Role for Rho-kinase in Rho-controlled Phospholipase D Stimulation by the m3 Muscarinic Acetylcholine Receptor" @default.
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