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• W2053144857 abstract "Dramatic transient changes resulting in a stellate morphology are induced in many cell types on treatment with agents that enhance intracellular cAMP levels. Thrombin fully protects cells from this inductive effect of cAMP through the thrombin receptor. The protective effect of thrombin was shown to be Rho-dependent. Clostridium botulinum C3 exoenzyme, which inactivates RhoA functions, abolished the ability of thrombin to protect cells from responding to increased cAMP levels. A constitutively activated RhoAV14 mutant protein also prevented cells from responding to cAMP. RhoA can be specifically phosphorylated at Ser-188 by the cAMP-activated protein kinase A (PKA). We demonstrate that RhoAV14A188, which cannot be phosphorylated by PKA in vitro, is more effective than RhoAV14 in preventing cells from responding to cAMP and in inducing actin stress fiber formation. This suggests that PKA phosphorylation of RhoA impairs its biological activity in vivo. ROKα, a RhoA-associated serine/threonine kinase can also prevent cells from responding to cAMP with shape changes. Phosphorylation of RhoA by PKA in vitro decreases the binding of RhoA to ROKα. These results indicate that RhoA and cAMP have antagonistic roles in regulating cellular morphology and suggest that cAMP-mediated down-regulation of RhoA binding to its effector ROKα may be involved in this antagonism. Dramatic transient changes resulting in a stellate morphology are induced in many cell types on treatment with agents that enhance intracellular cAMP levels. Thrombin fully protects cells from this inductive effect of cAMP through the thrombin receptor. The protective effect of thrombin was shown to be Rho-dependent. Clostridium botulinum C3 exoenzyme, which inactivates RhoA functions, abolished the ability of thrombin to protect cells from responding to increased cAMP levels. A constitutively activated RhoAV14 mutant protein also prevented cells from responding to cAMP. RhoA can be specifically phosphorylated at Ser-188 by the cAMP-activated protein kinase A (PKA). We demonstrate that RhoAV14A188, which cannot be phosphorylated by PKA in vitro, is more effective than RhoAV14 in preventing cells from responding to cAMP and in inducing actin stress fiber formation. This suggests that PKA phosphorylation of RhoA impairs its biological activity in vivo. ROKα, a RhoA-associated serine/threonine kinase can also prevent cells from responding to cAMP with shape changes. Phosphorylation of RhoA by PKA in vitro decreases the binding of RhoA to ROKα. These results indicate that RhoA and cAMP have antagonistic roles in regulating cellular morphology and suggest that cAMP-mediated down-regulation of RhoA binding to its effector ROKα may be involved in this antagonism. The mammalian Rho family of GTPases including RhoA, Rac1, and Cdc42 play pivotal roles in controlling many cellular functions including cell polarity, motility, proliferation, apoptosis, and cytokinesis (1Hall A. Annu. Rev. Cell Biol. 1994; 10: 31-54Crossref PubMed Scopus (768) Google Scholar). Morphological roles for these GTPases have mainly been established in fibroblasts. RhoA mediates the LPA 1The abbreviations used are: LPAlysophosphatidic acidBt2cAMPdibutyryl cAMPDTTdithiothreitolIBMX3-isobutyl-1-methylxanthineMES2-(N-morpholino)ethanesulfonic acidPKAprotein kinase AROKRho-binding kinaseFITCfluorescein isothiocyanateTRITCtetramethylrhodamine isothiocyanateGSTglutathioneS-transferase.-induced formation of stress fibers (2Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3843) Google Scholar). Rac1 mediates the effects of platelet-derived growth factor in regulating lamellipodia formation and membrane ruffling (3Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3084) Google Scholar). Cdc42 mediates the effects of bradykinin in the formation of filopodia in the cell periphery (4Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (883) Google Scholar). These morphological roles of Rho-family GTPases have also been demonstrated in other cell types including neuroblastoma cells (5Kozma R. Sarner S. Ahmed S. Lim L. Mol. Cell. Biol. 1997; 17: 1201-1211Crossref PubMed Scopus (535) Google Scholar), HeLa cells (6Leung T. Chen X.Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar, 7Manser E. Huang H.Y. Loo T.H. Chen X.Q. Dong J.M. Leung T. Lim L. Mol. Cell. Biol. 1997; 17: 1129-1143Crossref PubMed Google Scholar), and macrophages (8Allen W.E. Jones G.E. Pollard J.W. Ridley A.J. J. Cell Sci. 1997; 110: 707-720Crossref PubMed Google Scholar). However, in certain cells individual Rho-family GTPases may exert different morphological roles. For example, in KB cells, RhoA rather than Rac1 was shown to be involved in mediating membrane ruffling induced by hepatocyte growth factor and phorbol ester (9Nishiyama T. Sasaki T. Takaishi K. Kato M. Yaku H. Araki K. Matsuura Y. Takai Y. Mol. Cell. Biol. 1994; 14: 2447-2456Crossref PubMed Scopus (160) Google Scholar). An increasing number of Rho-family GTPase-associated proteins have been discovered within the last few years (for review see Ref. 10Lim L. Manser E. Leung T. Hall C. Eur. J. Biochem. 1996; 242: 171-185Crossref PubMed Scopus (273) Google Scholar) including serine/threonine kinases. With regard to morphology, these kinases appear to act as downstream effectors of the GTPases (11Lim L. Hall C. Monfries C. Semin. Cell Dev. Biol. 1996; 7: 699-706Crossref Scopus (17) Google Scholar). Among them, a Rho-binding serine/threonine kinase, ROK (12Ishizaki 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, 13Leung T. Manser E. Tan L. Lim L. J. Biol. Chem. 1995; 270: 29051-29054Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar, 14Matsui 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), promotes the formation of actin stress fibers and focal adhesion complexes (6Leung T. Chen X.Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar,15Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (951) Google Scholar). lysophosphatidic acid dibutyryl cAMP dithiothreitol 3-isobutyl-1-methylxanthine 2-(N-morpholino)ethanesulfonic acid protein kinase A Rho-binding kinase fluorescein isothiocyanate tetramethylrhodamine isothiocyanate glutathioneS-transferase. Neural cells, which can undergo differentiation in vitro,display marked changes in their morphology, some of which may reflect structural reorganization required for specific functional needs. For example, mature astrocytes in vivo have multiple elongated processes that are thought to be critical for their physiological functions. Astrocytes prepared from neonatal cerebral hemispheres or cerebella can undergo dramatic shape changes in tissue culture resulting in a stellated appearance similar to their in vivomorphology when exposed either to live neurons (16Hatten M.E. J. Cell Biol. 1985; 100: 384-396Crossref PubMed Scopus (507) Google Scholar) or cAMP analogues such as dibutyryl cAMP (Bt2cAMP) (17Baorto D.M. Mellado W. Shelanski M.L. J. Cell Biol. 1992; 117: 357-367Crossref PubMed Scopus (138) Google Scholar, 18Goldman J.E. Chiu F. Brain Res. 1984; 306: 85-95Crossref PubMed Scopus (107) Google Scholar). cAMP, as an intracellular second messenger, plays important roles in many aspects of nerve system functions. It has been shown recently that cAMP can induce the switching of directional turning of nerve growth cones in response to a gradient of diffusible factors, such as brain-derived growth factor, netrin-1, and acetylcholine (19Song H.-J. Ming G.-L. Poo M.-M. Nature. 1997; 388: 275-279Crossref PubMed Scopus (522) Google Scholar, 20Ming G.-L. Song H.-J. Berninger B. Holt C.E. Tessier-Lavigne M. Poo M.-M. Neuron. 1997; 19: 1225-1235Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar). The gradient of these molecules normally triggers an attractive turning response of growth cones. But when intracellular cAMP levels are reduced, the same gradient induces a repulsive turning response, which suggests that cAMP functions as a gate element in the pathfinding of neurite outgrowth. However, the mechanisms underlying these morphological changes are still poorly understood. Thrombin, a multi-functional protein including serine proteinase activity, has a remarkable ability to interact with a large variety of cell types and rapidly elicits a range of physiological responses. Thrombin can cause rapid neurite retraction in neuronal cells by activation of its cell surface receptors (21Jalink K. Moolenaar W.H. J. Cell Biol. 1992; 118: 411-419Crossref PubMed Scopus (168) Google Scholar, 22Suidan H.S. Stone S.R. Hemmings A. Monard D. Neuron. 1992; 8: 363-375Abstract Full Text PDF PubMed Scopus (233) Google Scholar). Interestingly, a comparable response has also been demonstrated in cells of glial lineage (23Cavanaugh K.P. Gurwitz D. Cunningham D.D. Bradshaw R.A. J. Neurochem. 1990; 54: 1735-1743Crossref PubMed Scopus (186) Google Scholar). The thrombin-stimulated neurite retraction in N1E-115 and PC12 neuronal-like cells has been shown to involve RhoA (24Jalink K. van Corven E.J. Hengeveld T. Morii N. Narumiya S. Moolenaar W.H. J. Cell Biol. 1994; 126: 801-810Crossref PubMed Scopus (577) Google Scholar). The link between thrombin receptor activation and Rho activation is as yet unknown. Many human neuroblastomas are tumors of early childhood derived from the neural crest, usually comprising a variety of cell types ranging from neuroblasts to melanocytes, glial cells and chondrocytes. Cell lines established from several of these tumors exhibit a similar diversity in their morphology and biochemical properties (25Ciccarone V. Spengler B.A. Meyers M.B. Biedler J.L. Ross R.A. Cancer Res. 1989; 49: 219-225PubMed Google Scholar). For example, both epithelial-like SH-EP cells with properties reminiscent of a melanocytic, Schwannian, and/or meningeal cell type and neuronal-like SH-SY cells are derived from the same human neuroblastoma SK-N-SH cell line (26Ross R.A. Spengler B.A. Biedler J.L. J. Natl. Cancer Inst. 1983; 71: 741-747PubMed Google Scholar). These two cell types appear to be capable of interconversion, a phenomenon called transdifferentiation (27Biedler J.L. Ross R.A. Meyers M.B. Rozen M. Spengler B.A. Proc. Am. Assoc. Cancer Res. 1984; 25: 41Google Scholar). We report here that treatment of SH-EP cells by forskolin, Bt2cAMP, or isoproterenol, which raise intracellular cAMP levels, caused rapid and transient cell morphological changes manifesting a stellate appearance. These treated cells round-up, leaving behind many of their processes. On the other hand, thrombin completely prevented SH-EP cells from responding to forskolin treatment morphologically. Microinjection of the constitutively activated RhoAV14 also effectively prevented cells from undergoing these cAMP-induced shape changes. The double mutant RhoAV14A188 with an alanine substituting the serine at position 188, which is specifically phosphorylated by the cAMP-activated protein kinase PKA in vitro (28Lang P. Resbert F. Delespine-Carmagnat M. Stancou R. Pouchelet M. Bertoglio J. EMBO J. 1996; 15: 510-519Crossref PubMed Scopus (481) Google Scholar), exerted a greater protective effect. ROKα also prevented cells from undergoing these cAMP-induced shape changes. ROKα was found to bind less of the RhoA upon phosphorylation of RhoA by PKA in vitro. Our results indicate that RhoA and cAMP have antagonistic roles in regulating cellular morphology and suggest that down-regulation of RhoA binding to effector ROKα may be involved in this antagonism. RhoA cDNA was cloned into pGEX-2T vector (Amersham Pharmacia Biotech) as pGEX-2TRhowt. PCR mediated mutagenesis was carried out to create pGEX-2TRhoA188, pGEX-2TRhoAV14, and pGEX-2TRhoAV14A188. RhoAA188 was also cloned into pGEX-2TK vector (Amersham Pharmacia Biotech), which contains an internal PKA phosphorylation site. The plasmids were sequenced before use for protein expression. The binding domain of ROKα was cloned into the pMAL vector (New England Biolabs) and C-terminally truncated ROKα cDNA was cloned into a mammalian expression vector, pXJ40HA to create construct pXJ40HAROKΔC as described previously (6Leung T. Chen X.Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar). cDNAs were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins using the pGEX-2T or pGEX-2TK vectors. Proteins were purified on glutathione-Sepharose beads as described (29Ridley A.J. Self A.J. Kasmi F. Paterson H.F. Hall A. Marshall C.J. Ellis C. EMBO J. 1993; 12: 5151-5160Crossref PubMed Scopus (199) Google Scholar). The proteins were released from the beads by thrombin cleavage and dialyzed against microinjection buffer (50 mm Tris (pH 7.5), 50 mm NaCl, 5 mm MgCl2, 0.1 mm DTT) after removing thrombin by benzamidine-Sepharose beads. The uncleaved GST-fusion proteins were also obtained as in vitro phosphorylation material by eluting from beads with reduced glutathione. Protein preparations showed essentially only one band on Coomassie blue-stained SDS-polyacrylamide gels. The protein concentration was determined by Bio-Rad protein assay reagent and Coomassie blue staining. SH-EP cells and SK-N-SH cells were grown on ethanol-washed glass, with a cross for relocation of microinjected cells, in Dulbecco's modified Eagle's medium with high glucose plus 10% fetal calf serum at 37 °C to subconfluence before treatment with various agents or microinjection. Recombinant and purified proteins were microinjected into the cytoplasm using an Eppendorf microinjector and Zeiss Axiovert microscope. Microinjection was performed within 10 min. Within this time frame, normally 30–60 cells could be successfully injected. To locate the injected cells, different concentrations of recombinant proteins were co-injected with rabbit IgG at 0.2–0.4 mg/ml. Clostridium botulinum C3 exoenzyme (Upstate Biotechnology Inc.) was co-injected at 0.16 mg/ml. After microinjection, cells were cultured in serum-free medium or serum-free medium containing 20 μm forskolin or other intracellular cAMP-elevating agents as indicated for 1 h before fixation. The plasmid pXJ40HAROKΔC (50 ng/ml) was injected into nuclei. 2 h after injection, cells were cultured in serum-free medium containing 20 μm forskolin for 1 h before fixation. Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline for 20 min, permeabilized with 0.2% Triton X-100 in phosphate-buffered saline for 10 min, and washed in phosphate-buffered saline for 10 min. Cells were then stained with 1:100 FITC-conjugated goat anti-rabbit IgG antibody (visualizing the injected cells) and 50 ng/ml TRITC-labeled phalloidin (visualizing filamentous actin) for 1 h at room temperature, washed in 0.1% Triton X-100 in phosphate-buffered saline two times for 5 min each. FITC-conjugated anti-hemagglutinin antibody was used to locate recombinant ROK-expressing cells. Cells were viewed on a Zeiss fluorescent microscope, and photographed with Kodak T-MAX 400 or Ektachrome 400 film, or viewed on an MRC 600 confocal imager. Phosphorylation of RhoA by PKAin vitro was carried out as described previously (28Lang P. Resbert F. Delespine-Carmagnat M. Stancou R. Pouchelet M. Bertoglio J. EMBO J. 1996; 15: 510-519Crossref PubMed Scopus (481) Google Scholar). Briefly, purified GST-fusion proteins were preloaded with either GDP or GTPγS before being bound to glutathione beads. Phosphorylation by PKA (10 units, catalytic subunit, Sigma) was carried out in a 25-μl final volume of phosphorylation buffer (50 mm Tris, pH 7.5, 10 mm MgCl2, 1 mm DTT, and 10 μCi [γ-32P]ATP (3000 Ci/mmol, Amersham Pharmacia Biotech)) for 30 min at 30 °C. The reaction was stopped by extensively washing with cold phosphorylation buffer. Proteins were eluted by boiling in SDS-polyacrylamide gel electrophoresis sample buffer and processed for SDS-polyacrylamide gel electrophoresis and autoradiography. 0.2 μg of each purified GST-fusion protein from pGEX-2TRhoAwt and pGEX-2TKRhoAA188was bound to glutathione beads and phosphorylated by PKA as described above in the presence of 10 μCi [γ-32P]ATP for GST-RhoAwt or 0.1 μCi [γ-32P]ATP for GST-K/RhoAA188, because the phosphorylation site provided by pGEX-2TK is a more preferable one than Ser-188 of RhoA. After washing with cold phosphorylation buffer once and cold exchange buffer (50 mm NaCl, 25 mm MES, pH 6.5, 1.25 mm EDTA, 1.25 mm DTT, and 0.6 mg/ml bovine serum albumin) twice, the beads were loaded with 10 μCi [35S]GTPγS in 20 μl of exchange buffer at room temperature for 5 min. The beads were then washed with 1 ml of stopping buffer (50 mm NaCl, 25 mm MES, pH 6.5, 1.25 mm MgCl2, 2.5 mm DTT, and 0.05% Triton X-100) for 5 times. The fusion proteins were eluted by reduced glutathione in 320 μl of stopping buffer. 20 μl of eluate were radioassayed using a dual label mode on a LKB 1219 Rackbata liquid scintillation counter. To eliminate overlapping of 35S and32P spectrums, the window 1 for 35S was narrowed to 5–450, and window 2 for 32P to 500–900. The remaining eluate was incubated with 5 μg of maltose-binding protein-fused ROK binding domain (6Leung T. Chen X.Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar), which had been prebound to 50 μl of maltose beads, in the presence of 10 mg/ml bovine serum albumin at 4 °C for 30 min. The beads were then washed with 1 ml of washing buffer (50 mm NaCl, 25 mm MES, pH 6.5, 5 mm MgCl2 and 0.05% Triton X-100) for 3 times and directly counted using a dual label mode. SH-EP cells cultivated in serum-free medium for 1 h manifested epithelia-like morphology (Fig. 1 A). The cells were flat and appeared to be tightly adherent to the culture matrix. Treatment with isoproterenol (0.1 μm), an agonist of the β-adrenergic receptor, resulted in a rapid morphological change. Flattened cell bodies started to round-up leaving many long processes (referred to as “cAMP phenotype”) as early as 10–15 min after addition of the drug. Maximal effects were observed within 1 h. The effects of isoproterenol resulted from increased intracellular cAMP levels, as it can activate adenylate cyclase activity via β-adrenergic receptors. Furthermore, treatment with either forskolin (20 μm), a direct adenylate cyclase activator, or dibutyryl cAMP (2 mm), a permeable cAMP analogue, elicited similar cell shape changes over the same time scale (Fig.1 B). IBMX (3-isobutyl-1-methylxanthine), a potent phosphodiesterase inhibitor that inhibits cAMP degradation, potentiated the effects of isoproterenol, forskolin, and dibutyryl-cAMP (data not shown). This change was transient because by 10–13 h, cells regained their original shape (Fig. 1 C). These reflattened cells became desensitized, because further treatment with forskolin caused no subsequent morphological change. These morphological effects did not occur in parental SK-N-SH cells or another subclonal line, SH-SY cells, which are neuronal-like in morphology. Co-treatment with thrombin (0.001 units/ml) completely blocked the morphological effects of forskolin; treated cells showed only slight contraction of the cell body when compared with controls (Fig. 2, A and B), which was also observed with thrombin treatment alone. Continuous exposure was not required for thrombin to exert its protective effect. No cAMP phenotype was observed when cells were transiently treated with thrombin for 10 min, followed by forskolin treatment in fresh medium for 1 h (data not shown). To rule out the possibility that the protective effects of thrombin were caused by nonspecific protease activity, another serine protease, trypsin, was also tested. At concentrations up to 0.1 units/ml, trypsin did not prevent cells from responding to forskolin treatment (data not shown). SFLL, a 14-amino acid peptide derived from residues 42–55 of the thrombin receptor, can duplicate all the actions of thrombin itself (30Vu T.K. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2680) Google Scholar). As shown in Fig. 2 C, inclusion of 10 μmSFLL in the medium completely blocked the morphological effect of forskolin. Thus thrombin antagonizes the effect of increased cAMP levels via activation of its receptor. Rho mediates LPA-induced stress fiber formation (2Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3843) Google Scholar). To test if thrombin can also induce actin stress fiber formation in SH-EP cells, cells were cultured in serum-free medium with or without 0.001 units/ml thrombin for 1 h. Control cells contained some thin stress fibers. Upon exposure to thrombin, large amounts of stress fibers were formed that were much thicker and denser (Fig. 2, D and E). This induction of stress fiber formation was observed as early as 10 min after thrombin treatment (data not shown). Microinjection of the Rho inhibitor C. botulinum C3 exoenzyme (0.16 mg/ml) completely blocked thrombin-activated stress fiber formation and dissolved existing stress fibers (Fig. 2, F and G). These results show Rho pathways to be implicated in thrombin-induced stress fiber formation in SH-EP cells and suggest an involvement of Rho in thrombin antagonism of cAMP effects. To test directly for RhoA involvement in the cAMP-induced cell shape changes, subconfluent SH-EP cells were microinjected with a constitutively activated RhoAV14 recombinant protein (1 mg/ml). Cells were then treated with 20 μm forskolin for 1 h before fixation. Injected cells were identified by immunostaining of co-injected rabbit IgG. RhoAV14effectively prevented cells from undergoing the cAMP-induced cell shape changes. As shown in Fig. 3, Aand B, the RhoAV14 injected cells maintained their shape after forskolin treatment. Massive stress fibers were formed (a typical Rho effect). The neighboring uninjected cells lost their stress fibers leaving diffused actin filaments around the cell body and in remaining processes. Co-injection of C3 exoenzyme with RhoAV14 completely abolished these Rho-mediated effects (Fig. 3, C and D). To quantify these effects, injected cells were identified and scored, based on their morphology after treatment with forskolin for 1 h. Those cells remaining in flattened cell shapes scored as no morphological change; those showing complete rounding up of the cell body with multiple processes scored as cAMP phenotype, and those with partially shrunk cell body bearing some branches scored as intermediate phenotype. As shown in Fig. 4, whereas >95% of RhoAV14-injected cells showed no morphological change upon forskolin treatment, less than 10% of control IgG-injected cells exhibited no morphological change. In RhoAV14 and C3 exoenzyme co-injected cells, more than 80% exhibited cAMP phenotype. These results imply that inactivation of RhoA may be one mechanism underlying the morphological changes induced by elevated intracellular cAMP levels. Elevated intracellular cAMP levels trigger signal-transduction pathways through activation of PKA, which phosphorylates many target proteins. One such target identified is RhoA, phosphorylated on Ser-188 (28Lang P. Resbert F. Delespine-Carmagnat M. Stancou R. Pouchelet M. Bertoglio J. EMBO J. 1996; 15: 510-519Crossref PubMed Scopus (481) Google Scholar), although the biological consequence of this RhoA phosphorylation in vivo was not reported. Recombinant GST-RhoAwt fusion protein was phosphorylated by the catalytic subunit of PKA in vitro(Fig. 5 A). However, the GST-RhoAA188 protein, with Ser-188 mutated to Ala-188 was not phosphorylated by PKA, confirming previous findings that PKA specifically phosphorylates Ser-188 of RhoA (28Lang P. Resbert F. Delespine-Carmagnat M. Stancou R. Pouchelet M. Bertoglio J. EMBO J. 1996; 15: 510-519Crossref PubMed Scopus (481) Google Scholar). Furthermore, although both can be phosphorylated by PKA in vitro, GDP-preloaded RhoA is a relatively better substrate than GTPγS-preloaded RhoA, which suggests that the phosphorylation of RhoA in vivo may be regulated depending on whether it is in an active or inactive state. To test the morphological consequences of RhoA phosphorylation, different concentrations of RhoAV14 or RhoAV14A188 proteins were microinjected into SH-EP cells which were then challenged with forskolin. If phosphorylation of RhoA does indeed down-regulate RhoA activity, loss of the Ser-188 phosphorylation site should enhance the protective effects of RhoA against forskolin (acting through cAMP activation of PKA). Injected cells were identified and scored as described before with the results expressed as the percentage of the injected cells with no morphological change (Fig. 5 B). The protective effect of RhoAV14 was concentration-dependent; the majority of RhoAV14-injected cells (>90%) remained flattened upon forskolin treatment at concentrations of 0.8–1 mg/ml, and <20% of RhoAV14-injected cells showed no morphological change below 0.6 mg/ml. At a concentration below 0.3 mg/ml, RhoAV14 was totally ineffective in protecting cells from the effects of forskolin. By contrast, more than 90% of cells were protected when injected with this concentration of RhoAV14A188. Even at concentrations as low as 0.18 mg/ml, 50% of RhoAV14A188-injected cells remained flattened. To confirm that the phosphorylation of RhoA itself also blocks stress fiber formation, SK-N-SH cells were used for microinjection experiment. Unlike SH-EP cells, upon forskolin treatment, SK-N-SH cells do not round-up but shrink slightly, thus changes in actin cytoskeletal structure are more easily observed. These SK-N-SH cells contain some basal stress fibers; these fibers are significantly reduced and often completely lost upon forskolin treatment (data not shown). Injection of RhoAV14 protein (0.6 mg/ml) potently induced stress fiber formation in control SK-N-SH cells (Fig.6 A) but not in forskolin-treated cells (Fig. 6 C). However, RhoAV14A188 protein, even when injected at lower concentrations (0.3 mg/ml), was able to induce stress fiber formation in forskolin-treated cells (Fig. 6 E). These results confirm that PKA antagonizes the biological activity of RhoA through phosphorylation of RhoA. ROKα was recently shown to direct the reorganization of the actin cytoskeleton with microinjection of an expression vector encoding ROKα, promoting formation of stress fibers and focal adhesion complexes in HeLa and fibroblast cells (6Leung T. Chen X.Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar). To test whether ROKα is also involved in cAMP-induced morphological changes in SH-EP cells, an expression vector encoding hyperactive ROKαΔC (6Leung T. Chen X.Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar) was microinjected into SH-EP cells, which were then challenged with forskolin. As shown in Fig.7, expression of ROKα stimulated actin stress fiber formation in SH-EP cells and protected cells against the morphological effects of forskolin (see neighboring cells). These results are consistent with ROKα acting downstream of RhoA because both proteins promoted stress fiber formation and protected against the effects of forskolin. Although phosphorylation of RhoA by PKA does not affect its binding of GTP (see Ref. 28Lang P. Resbert F. Delespine-Carmagnat M. Stancou R. Pouchelet M. Bertoglio J. EMBO J. 1996; 15: 510-519Crossref PubMed Scopus (481) Google Scholar; and data not shown), we wondered whether this could lead to down-regulation of its interaction with the effector ROKα. Because PKA phosphorylates RhoA poorly in vitro (Ref. 28Lang P. Resbert F. Delespine-Carmagnat M. Stancou R. Pouchelet M. Bertoglio J. EMBO J. 1996; 15: 510-519Crossref PubMed Scopus (481) Google Scholar; and data not shown), analysis of ROK binding of phosphorylated RhoA using radiolabeled GTP-RhoA as substrate (6Leung T. Chen X.Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar) will be confounded by the presence of larger amounts of unphosphorylated RhoA. We therefore used a double-labeling method to determine whether phosphorylation of RhoA affected its interaction with ROKα. GST-RhoAwt and control GST-K-RhoAA188 containing the polylinker PKA phosphorylation site were first phosphorylated by PKA with [γ-32P]ATP. The 32P-labeled proteins were subsequently loaded with [35S]GTPγS. 32P- and 35S-radioactivity associated with GST-RhoAwt or GST-K-RhoAA188 was simultaneously determined, before and after the proteins were bound to the immobilized binding domain of ROKα (Fig. 8). If PKA phosphorylation of RhoA inhibits its binding to ROKα, the32P/35S ratio should be lower in RhoA upon binding to ROKα beads, as less of the 32P-phosphorylated RhoA will contribute to the population of [35S]GTPγS-RhoA, which was bound to ROKα. On the other hand, if phosphorylation has no effect on binding of RhoA to ROKα, the 32P/35S ratio should be similar in RhoA before and after binding to ROKα. Fig. 8 shows that the32P/35S ratio in such an experiment decreased 50% when proteins containing [35S]GTPγS- and32P-labeled RhoAwt were bound to ROKα. In contrast, this ratio was essentially unaltered when proteins containing [35S]GTPγS- and 32P-labeled RhoAA188 was bound to ROKα. These results indicate that RhoA upon its phosphorylation by PKA may have decreased binding to ROKα. They also imply that cAMP-induced changes may be mediated through PKA down-regulation of RhoA binding to ROKα. Neuron and astroglial cell differentiation are accompanied by dramatic morphological changes over sustained periods in vivo and in cell culture. Similar morphological changes known as arborization or stellation can be rapidly induced in some animal cells, including astrocytes (17Baorto D.M. Mellado W. Shelanski M.L. J. Cell Biol. 1992; 117: 357-367Crossref PubMed Scopus (138) Google Scholar), mesangial cells (31Kreisberg J.I. Venkatachalam M.A. Patel P.Y. Kidney Int. 1984; 25: 874-879Abstract Full Text PDF PubMed Scopus (35) Google Scholar), fibroblasts (32Lamb N.J.C. Fernandez A. Conti M.A. Adelstein R. Glass D.B. Welch W.J. Feramisco J.R. J. Cell Biol. 1988; 106: 1955-1972Crossref PubMed Scopus (235) Google Scholar), and osteoblastic cells (33Egan J.J. Gronowicz G. Rodan G.A. J. Cell. Biochem. 1991; 45: 101-111Crossref PubMed Scopus (46) Google Scholar) in culture by increasing the intracellular cAMP level, which itself often acts as a differentiation agent. The diversity of cell types exhibiting similar responses to cAMP suggests its fundamental involvement in regulating cytoskeleton/membrane events. We report in this study that the epithelia-like SH-EP cell line can also undergo rapid and reversible cell shape changes, characterized by a stellate appearance, upon increasing intracellular cAMP levels. It has been reported that SH-EP cells can interconvert into neuronal-like cell types at low frequency (27Biedler J.L. Ross R.A. Meyers M.B. Rozen M. Spengler B.A. Proc. Am. Assoc. Cancer Res. 1984; 25: 41Google Scholar). Because the morphology of treated SH-EP cells that have undergone cAMP-induced cell shape changes resembles that of some neurons, we tested for the appearance of mRNA for neurofilament, a neuronal marker, which is absent in normal SH-EP cells. These cells did not contain neurofilament mRNA unlike its sibling neuronal-like SH-SY cells (data not shown). These results coupled with the reversion of morphological changes with time showed that cAMP was not promoting transdifferentiation but rather inducing transient effects in SH-EP cells. The cAMP-induced changes cannot be evoked when cells are grown in the presence of serum even as low as 1%, indicating that some factors in serum can antagonize the cAMP effect. Curiously, LPA, a serum activator of Rho (34Moolenaar W.H. Curr. Opin. Cell Biol. 1995; 7: 203-210Crossref PubMed Scopus (222) Google Scholar) did not prevent cells from responding to forskolin-induced changes in serum-starved SH-EP cells (data not shown). Our study shows another serum component, thrombin, to prevent forskolin and other intracellular cAMP-elevating agents from inducing morphological changes. In line with other studies (30Vu T.K. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2680) Google Scholar), our data suggests that thrombin functions through a specific receptor, as a peptide agonist of the thrombin receptor, SFLL exhibits the same protective effect. In SH-EP cells thrombin may induce stress fiber formation via a Rho pathway in a manner analogous to the LPA-induced pathway in fibroblasts. In support of this Rho mediation, we have found the protective ability of thrombin to antagonize forskolin action to be abrogated when SH-EP cells were first microinjected with C3 exoenzyme (data not shown). However, microinjection of C3 exoenzyme alone cannot mimic cAMP-induced morphological changes (Fig. 2, F andG; and data not shown), which indicates that inactivation of RhoA alone may not be sufficient for the full responsiveness of cAMP treatment. On the other hand, microinjection of constitutively activated RhoAV14 mimics the thrombin effect in blocking cAMP-induced morphological changes. Thrombin- or LPA-activated signal-transduction pathways and the cAMP second messenger system have been documented as playing opposing roles. For example, in neuronal cells, thrombin and LPA both rapidly trigger neurite retraction by activating their respective receptors (21Jalink K. Moolenaar W.H. J. Cell Biol. 1992; 118: 411-419Crossref PubMed Scopus (168) Google Scholar, 22Suidan H.S. Stone S.R. Hemmings A. Monard D. Neuron. 1992; 8: 363-375Abstract Full Text PDF PubMed Scopus (233) Google Scholar,35Jalink K. Eichholtz T. Postma F.R. van Corven E.J. Moolenaar W.H. Cell Growth Differ. 1993; 4: 247-255PubMed Google Scholar). Rho has been shown to be a component in these signal transduction pathways (24Jalink K. van Corven E.J. Hengeveld T. Morii N. Narumiya S. Moolenaar W.H. J. Cell Biol. 1994; 126: 801-810Crossref PubMed Scopus (577) Google Scholar). Conversely, cAMP can promote neurite outgrowth (36Rydel R.E. Greene L.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1257-1261Crossref PubMed Scopus (284) Google Scholar). Our results suggest that the cAMP effects in SH-EP cells may involve inactivation of Rho by PKA phosphorylation. However, other mechanisms by which cAMP negatively modulates RhoA activity may also exist. Recently, Laudanna et al. (37Laudanna C. Campbell J.J. Butcher E.C. J. Biol. Chem. 1997; 272: 24141-24144Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) have shown that pre-elevation of the intracellular level of cAMP prevents chemoattractant-stimulated RhoA GDP/GTP exchange in lymphoid cells. Thrombin exerts a contrary and protective effect by activation of the Rho pathway; how cross-talk between these two pathways occurs still remains to be established. The balance of cross-talk may be determined at least in part by the order of activation of RhoA and the activation of PKA in vivo. If RhoA is activated first, it may be more refractory to inactivation by phosphorylation, as in the case of blocking the cAMP effect by thrombin in SH-EP cells, because GTP-γ-S/RhoA is not as good a PKA substratein vitro as GDP/RhoA. If RhoA is phosphorylated in its GDP-form by PKA, it may be locked in the inactive state even if it can be converted to the GTP-form unless dephosphorylation takes place. Furthermore, PKA could also directly inhibit GTP exchange of RhoA in lymphoid cells (40). However, depending on the cell type, the balance of effects promoted by the two pathways can still be very different. In SH-EP cells, the thrombin-activated pathway dominates the cAMP-activated pathway at least in terms of its ability to maintain cell morphology. In contrast, increasing cAMP levels can protect against neurite retraction induced by LPA in neuronal-like PC12 cells (38Tigyi G. Fischer D.J. Sebok A. Marshall F. Dyer D.L. Miledi R. J. Neurochem. 1996; 66: 549-558Crossref PubMed Scopus (126) Google Scholar). During the course of this study, Lang et al. (28Lang P. Resbert F. Delespine-Carmagnat M. Stancou R. Pouchelet M. Bertoglio J. EMBO J. 1996; 15: 510-519Crossref PubMed Scopus (481) Google Scholar) demonstrated that recombinant RhoA is phosphorylated in vitro by PKA at Ser-188 and that this phosphorylation of RhoA increases its interaction with guanine-nucleotide dissociation inhibitor even in its GTP-bound state. They suggested that the translocation of phosphorylated GTP-Rho from membrane to cytosol by its binding to guanine-nucleotide dissociation inhibitor terminates RhoA signaling by preventing its interaction with downstream effectors, independently from its cycling from GTP to GDP. Using a phosphorylation-resistant RhoA mutant RhoAV14A188 and a nonresistant RhoA mutant RhoAV14, we have explored the biological consequences of RhoA phosphorylation in vivo. Our data show that the phosphorylation-resistant RhoA mutant is more potent than the nonresistant RhoA mutant in protecting SH-EP cells from forskolin-induced cell shape changes (Fig. 5). The activity of phosphorylation-resistant RhoA mutant to promote stress fiber formation is unaffected by forskolin at concentrations that effectively blocked this activity of the nonresistant RhoA mutant in SK-N-SH cells (Fig.6). Phosphorylation also impairs the binding of GTP-RhoA to its effector ROKα in vitro. Based on these observations, it is possible that PKA phosphorylation of RhoA may not only result in membrane removal and cytosolic sequestration of the GTP-Rho by guanine-nucleotide dissociation inhibitor as suggested by Lang et al. (28Lang P. Resbert F. Delespine-Carmagnat M. Stancou R. Pouchelet M. Bertoglio J. EMBO J. 1996; 15: 510-519Crossref PubMed Scopus (481) Google Scholar) but also reduces its interaction with ROKα. Both effects would result in a decreased translocation of ROK to membrane sites where it exerts its activity (6Leung T. Chen X.Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar, 10Lim L. Manser E. Leung T. Hall C. Eur. J. Biochem. 1996; 242: 171-185Crossref PubMed Scopus (273) Google Scholar, 13Leung T. Manser E. Tan L. Lim L. J. Biol. Chem. 1995; 270: 29051-29054Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar). The activation of Rho is a very rapid event with formation of the GTP-bound being accomplished within seconds of cellular exposure to factors (39Laudanna C. Campbell J.J. Butcher E.C. Science. 1996; 271: 981-983Crossref PubMed Scopus (434) Google Scholar). The multiple means of achieving inhibition of Rho outlined above and also by p190GAP (32Lamb N.J.C. Fernandez A. Conti M.A. Adelstein R. Glass D.B. Welch W.J. Feramisco J.R. J. Cell Biol. 1988; 106: 1955-1972Crossref PubMed Scopus (235) Google Scholar) infer that inhibition of this primary GTPase may be an important requirement for cytoskeletal reorganization to occur. Indeed growth cone development and neurite extension can occur in neuroblastomas and PC12 cells merely by inhibiting Rho (5Kozma R. Sarner S. Ahmed S. Lim L. Mol. Cell. Biol. 1997; 17: 1201-1211Crossref PubMed Scopus (535) Google Scholar, 24Jalink K. van Corven E.J. Hengeveld T. Morii N. Narumiya S. Moolenaar W.H. J. Cell Biol. 1994; 126: 801-810Crossref PubMed Scopus (577) Google Scholar, 38Tigyi G. Fischer D.J. Sebok A. Marshall F. Dyer D.L. Miledi R. J. Neurochem. 1996; 66: 549-558Crossref PubMed Scopus (126) Google Scholar). The SH-EP cells with their differential response to agents affecting morphology reported here provide an amenable vehicle for studying the morphological consequences of Rho activation and inactivation, and how cross-talk between Rho and PKA morphological pathway is achieved." @default.
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• W2053144857 title "cAMP-induced Morphological Changes Are Counteracted by the Activated RhoA Small GTPase and the Rho Kinase ROKα" @default.
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