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• W2024229206 abstract "Although muscarinic acetylcholine receptors (mAChR) regulate the activity of smooth muscle myosin, the effects of mAChR activation on cytoplasmic myosin have not been characterized. We found that activation of transfected human M3 mAChR induces the phosphorylation of myosin light chains (MLC) and the formation of myosin-containing stress fibers in Chinese hamster ovary (CHO-m3) cells. Direct activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) also induces myosin light chain phosphorylation and myosin reorganization in CHO-m3 cells. Conventional (α), novel (δ), and atypical (ι) PKC isoforms are activated by mAChR stimulation or PMA treatment in CHO-m3 cells, as indicated by PKC translocation or degradation. mAChR-mediated myosin reorganization is abolished by inhibiting conventional PKC isoforms with Go6976 (IC50 = 0.4 μm), calphostin C (IC50 = 2.4 μm), or chelerythrine (IC50 = 8.0 μm). Stable expression of dominant negative RhoAAsn-19 diminishes, but does not abolish, mAChR-mediated myosin reorganization in the CHO-m3 cells. Similarly, mAChR-mediated myosin reorganization is diminished, but not abolished, in CHO-m3 cells which are multi-nucleate due to inactivation of Rho with C3 exoenzyme. Expression of dominant negative RhoAAsn-19 or inactivation of RhoA with C3 exoenzyme does not affect PMA-induced myosin reorganization. These findings indicate that the PKC-mediated pathway of myosin reorganization (induced either by M3 mAChR activation or PMA treatment) can continue to operate even when RhoA activity is diminished in CHO-m3 cells. Conventional PKC isoforms and RhoA may participate in separate but parallel pathways induced by M3 mAChR activation to regulate cytoplasmic myosin. Changes in cytoplasmic myosin elicited by M3 mAChR activation may contribute to the unique ability of these receptors to regulate cell morphology, adhesion, and proliferation. Although muscarinic acetylcholine receptors (mAChR) regulate the activity of smooth muscle myosin, the effects of mAChR activation on cytoplasmic myosin have not been characterized. We found that activation of transfected human M3 mAChR induces the phosphorylation of myosin light chains (MLC) and the formation of myosin-containing stress fibers in Chinese hamster ovary (CHO-m3) cells. Direct activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) also induces myosin light chain phosphorylation and myosin reorganization in CHO-m3 cells. Conventional (α), novel (δ), and atypical (ι) PKC isoforms are activated by mAChR stimulation or PMA treatment in CHO-m3 cells, as indicated by PKC translocation or degradation. mAChR-mediated myosin reorganization is abolished by inhibiting conventional PKC isoforms with Go6976 (IC50 = 0.4 μm), calphostin C (IC50 = 2.4 μm), or chelerythrine (IC50 = 8.0 μm). Stable expression of dominant negative RhoAAsn-19 diminishes, but does not abolish, mAChR-mediated myosin reorganization in the CHO-m3 cells. Similarly, mAChR-mediated myosin reorganization is diminished, but not abolished, in CHO-m3 cells which are multi-nucleate due to inactivation of Rho with C3 exoenzyme. Expression of dominant negative RhoAAsn-19 or inactivation of RhoA with C3 exoenzyme does not affect PMA-induced myosin reorganization. These findings indicate that the PKC-mediated pathway of myosin reorganization (induced either by M3 mAChR activation or PMA treatment) can continue to operate even when RhoA activity is diminished in CHO-m3 cells. Conventional PKC isoforms and RhoA may participate in separate but parallel pathways induced by M3 mAChR activation to regulate cytoplasmic myosin. Changes in cytoplasmic myosin elicited by M3 mAChR activation may contribute to the unique ability of these receptors to regulate cell morphology, adhesion, and proliferation. Muscarinic acetylcholine receptors (mAChR) 1The abbreviations used are: mAChR, muscarinic acetylcholine receptor; BSA, bovine serum albumin; CaMKII, Ca2+/calmodulin-dependent protein kinase II; CHO, Chinese hamster ovary; FCS, fetal calf serum; HA, hemagglutinin; LPA, lysophosphatidic acid; MLC, myosin light chain; MLCK, myosin light chain kinase; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride.1The abbreviations used are: mAChR, muscarinic acetylcholine receptor; BSA, bovine serum albumin; CaMKII, Ca2+/calmodulin-dependent protein kinase II; CHO, Chinese hamster ovary; FCS, fetal calf serum; HA, hemagglutinin; LPA, lysophosphatidic acid; MLC, myosin light chain; MLCK, myosin light chain kinase; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride. are heterotrimeric G protein-coupled receptors that regulate contraction of smooth muscle. Multiple subtypes of mAChR that transduce different intracellular signals are often co-expressed in smooth muscle tissues (reviewed in Ref. 1Eglen R.M. Reddy H. Challiss R.A.J. Trends Pharmacol. Sci. 1994; 15: 114-119Abstract Full Text PDF PubMed Scopus (235) Google Scholar). The M1, M3, and M5 mAChR subtypes activate protein kinase C (PKC) by elevating intracellular Ca2+ and diacylglycerol. In contrast, the M2and M4 mAChR subtypes inhibit protein kinase A (PKA) by diminishing adenylyl cyclase activity (reviewed in Refs. 2Caulfield M.P. Pharmacol. Ther. 1993; 58: 319-379Crossref PubMed Scopus (1148) Google Scholar and 3Felder C.C. FASEB J. 1995; 9: 619-625Crossref PubMed Scopus (452) Google Scholar). Pharmacological studies indicate that M3 mAChR activation induces smooth muscle contraction, although activation of other co-expressed mAChR subtypes may modulate this response (reviewed in Ref. 1Eglen R.M. Reddy H. Challiss R.A.J. Trends Pharmacol. Sci. 1994; 15: 114-119Abstract Full Text PDF PubMed Scopus (235) Google Scholar). Dissection of the biochemical pathways involved in M3 mAChR-mediated contraction is complicated by the co-expression of multiple mAChR subtypes in many smooth muscle tissues.Contractile processes in non-muscle cells mimic those occurring in smooth muscle tissues, indicating the value of using non-muscle cells to investigate the biochemical pathways that regulate contraction (4Sellers J. Curr. Opin. Cell Biol. 1991; 3: 98-104Crossref PubMed Scopus (172) Google Scholar, 5Kolodney M.S. Elson E.L. J. Biol. Chem. 1993; 268: 23850-223855Abstract Full Text PDF PubMed Google Scholar, 6Goeckeler Z.M. Wysolmerski R.B. J. Cell Biol. 1995; 130: 613-627Crossref PubMed Scopus (376) Google Scholar, 7Chrzanowska-Wodnicka M. Burridge K. J. Cell Biol. 1996; 133: 1403-1415Crossref PubMed Scopus (1390) Google Scholar, 8Majumdar M. Seasholtz T.M. Goldstein D. de Lanerolle P. Brown J.H. J. Biol. Chem. 1998; 273: 10099-10106Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Phosphorylation of myosin light chains (MLC) in non-muscle cells causes the formation of myosin-containing stress fibers, which are contractile bundles of actin filaments associated with myosin II (4Sellers J. Curr. Opin. Cell Biol. 1991; 3: 98-104Crossref PubMed Scopus (172) Google Scholar, 5Kolodney M.S. Elson E.L. J. Biol. Chem. 1993; 268: 23850-223855Abstract Full Text PDF PubMed Google Scholar, 6Goeckeler Z.M. Wysolmerski R.B. J. Cell Biol. 1995; 130: 613-627Crossref PubMed Scopus (376) Google Scholar, 7Chrzanowska-Wodnicka M. Burridge K. J. Cell Biol. 1996; 133: 1403-1415Crossref PubMed Scopus (1390) Google Scholar, 8Majumdar M. Seasholtz T.M. Goldstein D. de Lanerolle P. Brown J.H. J. Biol. Chem. 1998; 273: 10099-10106Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Phosphorylation of MLC similarly increases actin-myosin interactions in smooth muscle cells, resulting in smooth muscle contraction (reviewed in Refs. 9Somylo A.P. Somylo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1724) Google Scholar, 10Takuwa Y. Jpn. Heart J. 1996; 37: 793-813Crossref PubMed Scopus (29) Google Scholar, 11Savineau J.P. Marthan R. Fundam. Clin. Pharmacol. 1997; 11: 289-299Crossref PubMed Scopus (65) Google Scholar, 12Narumiya S. Ishizaki T. Watanabe N. FEBS Lett. 1997; 410: 68-72Crossref PubMed Scopus (328) Google Scholar). In addition to smooth muscle contraction, many fundamental cellular processes such as adhesion, migration, and division depend upon the interaction of myosin with actin in contractile filaments (reviewed in Ref. 12Narumiya S. Ishizaki T. Watanabe N. FEBS Lett. 1997; 410: 68-72Crossref PubMed Scopus (328) Google Scholar). Activation of mAChR may affect these fundamental processes by altering myosin activity in non-muscle cells as it does in smooth muscle. Although mAChR activation induces MLC phosphorylation and subsequent contraction in smooth muscle cells (13Abdel-Latif A.A. Howe P.H. Akhtar R.A. Prog. Clin. Biol. Res. 1987; 249: 119-132PubMed Google Scholar, 14Colburn J.C. Michnoff C.H. Hsu L.-C. Slaughter C.A. Kamm K.E. Stull J.T. J. Biol. Chem. 1988; 263: 19166-19173Abstract Full Text PDF PubMed Google Scholar, 15Taylor D.A. Stull J.T. J. Biol. Chem. 1988; 263: 14456-14462Abstract Full Text PDF PubMed Google Scholar, 16Kamm K.E. Hsu L.-C. Kubota Y. Stull J.T. J. Biol. Chem. 1989; 264: 21223-21229Abstract Full Text PDF PubMed Google Scholar, 17Lucius C. Arner A. Steusloff A. Troschka M. Hofmann F. Aktories K. Pfitzer G. J. Physiol. (Lond.). 1998; 506: 83-93Crossref Scopus (70) Google Scholar), the ability of mAChR to regulate MLC phosphorylation and myosin organization in non-muscle cells has not been reported.We investigated the ability of transfected human mAChR subtypes to regulate myosin organization in Chinese hamster ovary (CHO) cells. Activation of transfected M3 mAChR induces MLC phosphorylation and causes myosin-containing stress fibers to form in CHO cells. The involvement of PKC in these events is indicated by our findings that 1) direct activation of PKC with phorbol esters induces MLC phosphorylation and myosin reorganization in CHO cells, 2) specific PKC antagonists inhibit M3 mAChR-mediated myosin reorganization, and 3) activation of transfected M1 but not M2 mAChR subtypes also induces the formation of myosin-containing stress fibers, demonstrating that only mAChR subtypes that stimulate PKC activity induce myosin reorganization. The participation of myosin light chain kinase (MLCK) and RhoA in mAChR-mediated myosin reorganization was also investigated, since these proteins regulate contractile processes in other systems (reviewed in Refs. 9Somylo A.P. Somylo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1724) Google Scholar, 10Takuwa Y. Jpn. Heart J. 1996; 37: 793-813Crossref PubMed Scopus (29) Google Scholar, 11Savineau J.P. Marthan R. Fundam. Clin. Pharmacol. 1997; 11: 289-299Crossref PubMed Scopus (65) Google Scholar, 12Narumiya S. Ishizaki T. Watanabe N. FEBS Lett. 1997; 410: 68-72Crossref PubMed Scopus (328) Google Scholar). We found that MLCK antagonists inhibit mAChR-mediated myosin reorganization but only at antagonist concentrations that may affect PKC. Stable expression of dominant negative RhoAAsn-19 or inactivation of Rho with C3 exoenzyme lessens mAChR-mediated myosin reorganization but does not abolish it.This study demonstrates that M3 mAChR activation significantly affects myosin organization in non-muscle cells. Our findings indicate that M3 mAChR activation induces cytoplasmic myosin reorganization by both PKC- and Rho-dependent mechanisms. These receptor-mediated changes in cytoplasmic myosin may contribute to the unique ability of M3 mAChR to regulate the adhesion (18Williams C.L. Hayes V.Y. Hummel A.M. Tarara J.E. Halsey T.J. J. Cell Biol. 1993; 121: 643-654Crossref PubMed Scopus (91) Google Scholar, 19Quigley R.L. Shafer S.H. Williams C.L. Chest. 1998; 114: 839-846Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 20Shafer S.H. Puhl H. Phelps S.H. Williams C.L. Exp. Cell Res. 1999; 248: 148-159Crossref PubMed Scopus (18) Google Scholar) and morphology (21Felder C.C. MacArthur L. Ma A.L. Gusovsky F. Kohn E.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1706-1710Crossref PubMed Scopus (45) Google Scholar, 22Singer-Lahat D. Ma A.L. Felder C.C. Biochem. Pharmacol. 1996; 51: 495-502Crossref PubMed Scopus (8) Google Scholar) of non-muscle cells.DISCUSSIONThis study demonstrates that M1 or M3mAChR activation profoundly alters myosin organization in CHO cells. Conventional PKC isoforms and Rho proteins participate in the mAChR-mediated formation of myosin stress fibers, as depicted in the model shown in Fig. 10. Similar pathways of mAChR-mediated myosin reorganization occur in CHO cells and smooth muscle cells, although some differences exist. Comparing these pathways provides insight into the mAChR-mediated mechanisms controlling myosin organization.Role of PKC in mAChR-mediated Myosin ReorganizationPKC participates in mAChR-mediated myosin reorganization in CHO cells. This conclusion is supported by our finding that M3 mAChR stimulation or PMA treatment activates several PKC isoforms and induces myosin stress fiber formation in these cells. Our finding that myosin reorganization is induced by the M1 but not the M2 mAChR subtype also supports this conclusion, since the M1 but not the M2 mAChR subtype activates PKC (2Caulfield M.P. Pharmacol. Ther. 1993; 58: 319-379Crossref PubMed Scopus (1148) Google Scholar, 3Felder C.C. FASEB J. 1995; 9: 619-625Crossref PubMed Scopus (452) Google Scholar).The effects of the PKC antagonists Go6976, calphostin C, and chelerythrine provide compelling evidence that conventional PKC isoforms are required for myosin reorganization. Both mAChR-mediated stress fiber formation and cell spreading are inhibited by calphostin C or chelerythrine, which antagonize conventional and novel PKC isoforms (34Tamaoki T. Methods Enzymol. 1991; 201: 340-347Crossref PubMed Scopus (329) Google Scholar, 35Herbert J.M. Augereau J.M. Gleye J. Maffrand J.P. Biochem. Biophys. Res. Commun. 1990; 172: 993-999Crossref PubMed Scopus (1184) Google Scholar). In contrast, only mAChR-mediated myosin reorganization is inhibited by Go6976, which specifically antagonizes conventional PKC isoforms (36Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Kochs G. Hug H. Marme D. Schachtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar). It is generally believed that PKC activation is required for cell spreading, and other signals are required for cytoskeletal reorganization (45Jarvinen M. Ylanne J. Vartio T. Virtanen I. Eur. J. Cell Biol. 1987; 44: 238-246PubMed Google Scholar, 46Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 47Defilippi P. Venturino M. Gulino D. Duperray A. Boquet P. Fiorentini C. Volpe G. Palmieri M. Silengo L. Tarone G. J. Biol. Chem. 1997; 272: 21726-21734Crossref PubMed Scopus (84) Google Scholar, 48Slack B.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7281-7286Crossref PubMed Scopus (55) Google Scholar, 49Somers C.E. Mosher D.F. J. Biol. Chem. 1993; 268: 22277-22280Abstract Full Text PDF PubMed Google Scholar, 50Vuori K. Ruoslahti E. J. Biol. Chem. 1993; 268: 21459-21462Abstract Full Text PDF PubMed Google Scholar, 51Mogi A. Hatai M. Soga H. Takenoshita S. Nagamachi Y. Fujimoto Y. Yamamoto T. Yokota J. Yaoi Y. FEBS Lett. 1995; 373: 135-140Crossref PubMed Scopus (16) Google Scholar). Our findings suggest that while the novel PKC isoforms are needed for cell spreading, the conventional PKC isoforms are needed for myosin reorganization.The concentrations of Go6976, calphostin C, or chelerythrine which inhibit myosin reorganization in CHO cells are approximately 10–70-fold higher than those that inhibit PKC in vitro(34Tamaoki T. Methods Enzymol. 1991; 201: 340-347Crossref PubMed Scopus (329) Google Scholar, 35Herbert J.M. Augereau J.M. Gleye J. Maffrand J.P. Biochem. Biophys. Res. Commun. 1990; 172: 993-999Crossref PubMed Scopus (1184) Google Scholar, 36Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Kochs G. Hug H. Marme D. Schachtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar). Higher concentrations of these drugs may be needed in vivo for the antagonists to cross the cell membrane and gain access to intracellular PKC, in contrast to in vitro studies in which the antagonists interact directly with purified PKC. Despite this requirement for higher drug concentrations in vivo, these antagonists still inhibit myosin reorganization more effectivelyin vivo than they inhibit other kinases such as PKA in vitro. This finding indicates that these antagonists inhibit myosin reorganization by inactivating PKC.The ability of PKC to regulate myosin organization in CHO cells is consistent with the effects of PKC activation on smooth muscle myosin. Activation of PKC with phorbol esters enhances MLC phosphorylation in smooth muscle cells and induces sustained contraction of different smooth muscle tissues (reviewed in Ref. 10Takuwa Y. Jpn. Heart J. 1996; 37: 793-813Crossref PubMed Scopus (29) Google Scholar). Phosphorylation of MLC increases actin-myosin interactions by inducing the formation of bipolar myosin filaments and exposing actin-binding sites on myosin (reviewed in Refs. 9Somylo A.P. Somylo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1724) Google Scholar, 10Takuwa Y. Jpn. Heart J. 1996; 37: 793-813Crossref PubMed Scopus (29) Google Scholar, 11Savineau J.P. Marthan R. Fundam. Clin. Pharmacol. 1997; 11: 289-299Crossref PubMed Scopus (65) Google Scholar, 12Narumiya S. Ishizaki T. Watanabe N. FEBS Lett. 1997; 410: 68-72Crossref PubMed Scopus (328) Google Scholar). PKC can directly phosphorylate MLC (52Bengur A.B. Robinson E.A. Apella E. Sellers J.R. J. Biol. Chem. 1987; 262: 7613-7617Abstract Full Text PDF PubMed Google Scholar, 53Ikebe M. Hartshorne D.J. Elzinga M. J. Biol. Chem. 1987; 262: 9569-9573Abstract Full Text PDF PubMed Google Scholar) or enhance MLC phosphorylation by inhibiting MLC phosphatase (54Masuo M. Reardon S. Ikebe M. Kitazawa T. J. Gen. Physiol. 1994; 104: 265-286Crossref PubMed Scopus (167) Google Scholar). It is believed that smooth muscle contraction induced by phorbol esters involves the PKC-dependent inhibition of MLC phosphatase, rather than direct phosphorylation of MLC by PKC (reviewed in Refs.9Somylo A.P. Somylo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1724) Google Scholar, 10Takuwa Y. Jpn. Heart J. 1996; 37: 793-813Crossref PubMed Scopus (29) Google Scholar, 11Savineau J.P. Marthan R. Fundam. Clin. Pharmacol. 1997; 11: 289-299Crossref PubMed Scopus (65) Google Scholar). PKC activation may similarly enhance MLC phosphorylation in CHO cells by inhibiting MLC dephosphorylation, as depicted in Fig. 10.Although PKC is required for carbachol-induced myosin reorganization in CHO-m3 cells, the role of PKC in the mAChR-mediated regulation of smooth muscle myosin is less clear. PKC antagonists inhibit carbachol-induced contractions in some types of smooth muscle (55Satoh M. Hayasaka M. Horiuchi K. Takayanagi I. Gen. Pharmacol. 1998; 30: 103-107Crossref PubMed Scopus (5) Google Scholar) but not in others (56Bremerich D.H. Warner D.O. Lorenz R.R. Shumway R. Jones K.A. Am. J. Physiol. 1997; 273: L775-L781PubMed Google Scholar). These variable responses may be due to the expression of different mAChR subtypes or PKC isoforms by different types of smooth muscle.It was previously shown that PKC activation induces or enhances the formation of actin-containing stress fibers in some types of non-muscle cells (45Jarvinen M. Ylanne J. Vartio T. Virtanen I. Eur. J. Cell Biol. 1987; 44: 238-246PubMed Google Scholar, 47Defilippi P. Venturino M. Gulino D. Duperray A. Boquet P. Fiorentini C. Volpe G. Palmieri M. Silengo L. Tarone G. J. Biol. Chem. 1997; 272: 21726-21734Crossref PubMed Scopus (84) Google Scholar, 57Woods A. Couchman J.R. J. Cell Sc i. 1992; 101: 277-290PubMed Google Scholar) but not in others (58Ridley A.J. Hall A. EMBO J. 1994; 13: 2600-2610Crossref PubMed Scopus (439) Google Scholar). These results indicate that PKC activation has cell type-specific effects on stress fiber formation. These specific effects may be due to altered PKC isoform expression or dissimilarities in PKC-mediated signaling pathways among different cell types.Activation of M3 mAChR induces greater MLC phosphorylation and myosin reorganization than does treatment with PMA in CHO cells. We found that PKC-α remains at the junctions of carbachol-treated cells longer than it does in PMA-treated cells, indicating that mAChR stimulation and PMA treatment have different effects on PKC-α. These differences may contribute to the greater stress fiber formation induced by carbachol compared with PMA, as well as the sustained elongation of CHO-m3 cells which occurs during prolonged carbachol exposure (Fig. 1, panel D, and Fig. 4 a, panel D) (21Felder C.C. MacArthur L. Ma A.L. Gusovsky F. Kohn E.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1706-1710Crossref PubMed Scopus (45) Google Scholar, 22Singer-Lahat D. Ma A.L. Felder C.C. Biochem. Pharmacol. 1996; 51: 495-502Crossref PubMed Scopus (8) Google Scholar).Role of RhoA in mAChR-mediated Myosin ReorganizationIncreased stress fiber formation in cells expressing constitutively active RhoAVal-14 indicates that active RhoA contributes to myosin reorganization in CHO-m3 cells. However, active RhoA is not essential for mAChR-mediated myosin reorganization in CHO-m3 cells. This conclusion is supported by our finding that carbachol-induced stress fiber formation is diminished but not abolished in CHO-m3 cells expressing dominant negative RhoAAsn-19 or treated with C3 exoenzyme. Expression of dominant negative RhoAAsn-19 may specifically inhibit RhoA activity because dominant negative RhoAAsn-19 competitively interacts with RhoA regulatory proteins. In contrast, C3 exoenzyme can ADP-ribosylate several forms of Rho, including RhoA and RhoB (59Mohr C. Koch G. Just I. Aktories K. FEBS Lett. 1992; 297: 95-99Crossref PubMed Scopus (21) Google Scholar), resulting in potentially greater Rho inactivation than that produced by expressing dominant negative RhoAAsn-19. Consistent with these possibilities, we found that the morphology of CHO-m3 cells is altered more drastically by C3 exoenzyme than by expression of dominant negative RhoAAsn-19.Treatment with C3 exoenzyme causes CHO-m3 cells to become multi-nucleate, which is an indication of Rho inactivation (60Rubin E.J. Gill D.M. Boquet P. Popoff M.R. Mol. Cell. Biol. 1988; 8: 418-426Crossref PubMed Scopus (233) Google Scholar, 61Mabuchi I. Hamaguchi Y. Fujimoto H. Morii N. Mishima M. Narumiya S. Zygote. 1993; 1: 325-331Crossref PubMed Scopus (213) Google Scholar, 62Kishi K. Sasaki T. Kuroda S. Itoh T. Takai Y. J. Cell Biol. 1993; 120: 1187-1195Crossref PubMed Scopus (308) Google Scholar). If Rho is inactive in dividing cells, the actomyosin contractile ring at the cleavage furrow does not function properly and cytokinesis is inhibited, resulting in multi-nucleate cells (61Mabuchi I. Hamaguchi Y. Fujimoto H. Morii N. Mishima M. Narumiya S. Zygote. 1993; 1: 325-331Crossref PubMed Scopus (213) Google Scholar, 62Kishi K. Sasaki T. Kuroda S. Itoh T. Takai Y. J. Cell Biol. 1993; 120: 1187-1195Crossref PubMed Scopus (308) Google Scholar). The ability of carbachol to induce stress fiber formation in multi-nucleate, C3 exoenzyme-treated CHO-m3 cells provides strong evidence that mAChR-mediated stress fiber formation still occurs even when Rho is inactive.Although Rho is apparently not essential for stress fiber formation induced by mAChR activation, Rho may be required for stress fiber formation induced by other agonists. Rho must be active for bombesin or lysophosphatidic acid (LPA) to induce stress fiber formation in serum-starved Swiss 3T3 cells (58Ridley A.J. Hall A. EMBO J. 1994; 13: 2600-2610Crossref PubMed Scopus (439) Google Scholar, 63Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3797) Google Scholar). Bombesin- or LPA-dependent stress fiber formation is significantly diminished by 27 μm genistein and is completely abolished by 110 μm genistein, indicating that a tyrosine kinase is required for stress fiber formation induced by these agonists (58Ridley A.J. Hall A. EMBO J. 1994; 13: 2600-2610Crossref PubMed Scopus (439) Google Scholar). In contrast, we found that genistein concentrations up to 180 μm do not alter mAChR-mediated stress fiber formation (data not shown). These findings indicate that different Rho-mediated signaling pathways leading to stress fiber formation are induced by bombesin or LPA in serum-starved Swiss 3T3 cells and by mAChR activation in exponentially proliferating CHO-m3 cells.Rho inactivation diminishes the agonist-induced contraction of smooth muscle (17Lucius C. Arner A. 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These and other findings support the model that Rho participates in contraction by enhancing MLC phosphorylation (reviewed in Refs. 10Takuwa Y. Jpn. Heart J. 1996; 37: 793-813Crossref PubMed Scopus (29) Google Scholar, 11Savineau J.P. Marthan R. Fundam. Clin. Pharmacol. 1997; 11: 289-299Crossref PubMed Scopus (65) Google Scholar, 12Narumiya S. Ishizaki T. Watanabe N. FEBS Lett. 1997; 410: 68-72Crossref PubMed Scopus (328) Google Scholar). Active Rho proteins can enhance MLC phosphorylation by inhibiting MLC phosphatase (69Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2423) Google Scholar) or by activating Rho kinase, which directly phosphorylates MLC (70Amano 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 (1663) Google Scholar, 71Kureishi Y. Kobayashi S. Amano M. Kimura K. Kanaide H. Nakano T. Kaibuchi K. Ito M. J. Biol. Chem. 1997; 272: 12257-12260Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). We are investigating the possibility that Rho proteins similarly regulate myosin organization in CHO cells by altering MLC phosphorylation, as depicted in Fig. 10. This possibility is supported by studies demonstrating that Rho inactivation diminishes MLC phosphorylation in non-muscle cells (8Majumdar M. Seasholtz T.M. Goldstein D. de Lanerolle P. Brown J.H. J. Biol. Chem. 1998; 273: 10099-10106Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 72Kreisberg J.I. Ghosh-Choudhury N. Radnik R.A. Schwartz M.A. Am. J. Physiol. 1997; 251: C505-C511Crossref Google Scholar).Rho inactivation in CHO-m3 cells does not diminish PMA-induced myosin reorganization, even though it diminishes mAChR-mediated myosin reorganization. These findings are consistent with a report that Rho inactivation does not affect phorbol ester-induced contraction of cerebrovascular smooth muscle but inhibits serotonin-induced contraction of the same tissue (64Akopov S.E. Zhang L. Pearce W.J. Am. J. Physiol. 1998; 275: H930-H939PubMed Google Scholar). These results may occur because Rho and PKC participate in separate signaling pathways to regulate myosin organization, as depicted in Fig. 10. According to this model, PKC induces myosin reorganization independently of Rho. This model explains why Rho inactivation does not affect PMA-induced myosin reorganization but diminishes mAChR-mediated myosin reorganization. This model also explains why Rho inactivation does not completely abolish mAChR-mediated stress fiber formation; mAChR-mediated activation of PKC induces myosin reorganization even when Rho is inactive.We found that PKC antagonists completely abolish mAChR-mediated stress fiber formation. This finding indicates that PKC inactivation inhibits both Rho- and PKC-dependent myosin reorganization induced by mAChR stimulation. This result may occur because PKC inactivation inhibits mAChR-mediated signaling to Rho. PKC may act in parallel with sever" @default.
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• W2024229206 date "1999-06-01" @default.
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• W2024229206 title "M3 Muscarinic Acetylcholine Receptors Regulate Cytoplasmic Myosin by a Process Involving RhoA and Requiring Conventional Protein Kinase C Isoforms" @default.
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