Gα13 Requires Palmitoylation for Plasma Membrane Localization, Rho-dependent Signaling, and Promotion of p115-RhoGEF Membrane Binding (2000)

Most heterotrimeric G protein α subunits are covalently modified by palmitate attached to one or more N-terminal cysteine residues. Although a wide variety of proteins undergo palmitoylation, the role of this fatty acid modification in G protein signaling is not well understood. Thus, we examined the role of palmitoylation of α13, a G protein α subunit that regulates many pathways involved in cell growth. Both N-terminal cysteines at positions 14 and 18 were required for palmitoylation. Mutant α13, in which both cysteines were changed to serines, failed to localize to plasma membranes in transfected cells and failed to activate Rho-dependent serum response factor-mediated transcription and actin stress fiber formation. However, nonpalmitoylated, cysteine to serine mutant α13retained the ability to co-immunoprecipitate with a direct effector, p115-RhoGEF. Finally, we report the novel observation that activated α13 induces a redistribution of p115-RhoGEF from the cytoplasm to plasma membranes, but non-palmitoylated mutants of α13 fail to cause p115-RhoGEF translocation. These findings identify palmitoylation of α13 as critical for its proper membrane localization and signaling and provide insight into the mechanism of activation of Rho-dependent signaling pathways by α13. Most heterotrimeric G protein α subunits are covalently modified by palmitate attached to one or more N-terminal cysteine residues. Although a wide variety of proteins undergo palmitoylation, the role of this fatty acid modification in G protein signaling is not well understood. Thus, we examined the role of palmitoylation of α13, a G protein α subunit that regulates many pathways involved in cell growth. Both N-terminal cysteines at positions 14 and 18 were required for palmitoylation. Mutant α13, in which both cysteines were changed to serines, failed to localize to plasma membranes in transfected cells and failed to activate Rho-dependent serum response factor-mediated transcription and actin stress fiber formation. However, nonpalmitoylated, cysteine to serine mutant α13retained the ability to co-immunoprecipitate with a direct effector, p115-RhoGEF. Finally, we report the novel observation that activated α13 induces a redistribution of p115-RhoGEF from the cytoplasm to plasma membranes, but non-palmitoylated mutants of α13 fail to cause p115-RhoGEF translocation. These findings identify palmitoylation of α13 as critical for its proper membrane localization and signaling and provide insight into the mechanism of activation of Rho-dependent signaling pathways by α13. guanine nucleotide-binding regulatory protein hemagglutinin serum response factor regulator of G protein signaling cysteine to serine phosphate-buffered saline Heterotrimeric (αβγ) G proteins1 act as molecular switches that transmit signals from heptahelical G protein-coupled receptors at the cell surface to intracellular effectors. G proteins regulate the activity of a variety of effector enzymes that generate second messengers or directly regulate other signaling proteins. Many G protein pathways propagate a signal to the nucleus, where they ultimately regulate gene expression. Accurate signal transduction in such pathways depends upon proper subcellular localization and, often, regulated changes in localization of constituent proteins. G protein α subunits (Gα) and βγ subunits (Gβγ) are often found tightly associated with cellular membranes, where receptors and many effectors also reside. Membrane binding is mediated, at least in part, by covalently attached lipids (1.Dunphy J.T. Linder M.E. Biochim. Biophys. Acta. 1998; 1436: 245-261Crossref PubMed Scopus (312) Google Scholar, 2.Wedegaertner P.B. Biol. Signals Receptors. 1998; 7: 125-135Crossref PubMed Scopus (69) Google Scholar). All Gα are modified at their N termini by myristate and/or palmitate (3.Buss J.E. Mumby S.M. Casey P.J. Gilman A.G. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7493-7497Crossref PubMed Scopus (198) Google Scholar, 4.Jones T.L. Simonds W.F. Merendino J.J. Brann M.R. Spiegel A.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 568-572Crossref PubMed Scopus (163) Google Scholar, 5.Mumby S.M. Heukeroth R.O. Gordon J.I. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 728-732Crossref PubMed Scopus (210) Google Scholar, 6.Parenti M. Vigano M.A. Newman C.M.H. Milligan G. Magee A.I. Biochem. J. 1993; 291: 349-353Crossref PubMed Scopus (123) Google Scholar, 7.Linder M.E. Middleton P. Hepler J.R. Taussig R. Gilman A.G. Mumby S.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3675-3679Crossref PubMed Scopus (291) Google Scholar). Myristoylation is a co-translational attachment of 14-carbon myristate to glycine at position 2 via an irreversible amide linkage. Palmitoylation, on the other hand, is a reversible post-translational attachment of 16-carbon palmitate via a thioester bond to cysteine residues. Gα can be divided into four families, αs, αi, αq, and α12, based on amino acid identity, and they can also be categorized based on the type and number of N-terminal fatty acid acylations. Gα can be myristoylated (e.g. αt), both myristoylated and palmitoylated (e.g. αi, αo, αz), singly palmitoylated (e.g.αs, α12), or dually palmitoylated (e.g. αq). The reversible nature of palmitoylation makes it a particularly interesting lipid modification, and, indeed, regulation of palmitoylation has been observed for several Gα (8.Degtyarev M.Y. Spiegel A.M. Jones T.L.Z. J. Biol. Chem. 1993; 268: 23769-23772Abstract Full Text PDF PubMed Google Scholar, 9.Wedegaertner P.B. Bourne H.R. Cell. 1994; 77: 1063-1070Abstract Full Text PDF PubMed Scopus (304) Google Scholar, 10.Mumby S.M. Kleuss C. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2800-2804Crossref PubMed Scopus (219) Google Scholar, 11.Stanislaus D. Janovick J.A. Brothers S. Conn P.M. Mol. Endocrinol. 1997; 11: 738-746PubMed Google Scholar, 12.Bhamre S. Wang H.Y. Friedman E. J. Pharmacol. Exp. Ther. 1998; 286: 1482-1489PubMed Google Scholar). In addition, palmitoylation, rather than myristoylation, may dictate specific targeting to plasma membranes, as opposed to intracellular membranes (13.Schroeder H. Leventis R. Shahinian P.A. Silvius J.R. J. Cell Biol. 1996; 134: 647-660Crossref PubMed Scopus (66) Google Scholar, 14.Wolven A. Okamura H. Rosenblatt Y. Resh M.D. Mol. Biol. Cell. 1997; 8: 1159-1173Crossref PubMed Scopus (146) Google Scholar, 15.Morales J. Fishburn C.S. Wilson P.T. Bourne H.R. Mol. Biol. Cell. 1998; 9: 1-14Crossref PubMed Scopus (80) Google Scholar). Despite much effort, very little is known regarding the biochemistry of and potential enzymes involved in palmitoylation (16.Duncan J.A. Gilman A.G. J. Biol. Chem. 1996; 271: 23594-23600Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 17.Dunphy J.T. Greentree W.K. Manahan C.L. Linder M.E. J. Biol. Chem. 1996; 271: 7154-7159Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 18.Das A.K. Dasgupta B. Bhattacharyya R. Basu J. J. Biol. Chem. 1997; 272: 11021-11025Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 19.Camp L.A. Verkruyse L.A. Afendis S.J. Slaughter C.A. Hofmann S.L. J. Biol. Chem. 1994; 269: 23212-23219Abstract Full Text PDF PubMed Google Scholar, 20.Duncan J.A. Gilman A.G. J. Biol. Chem. 1998; 273: 15830-15837Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 21.Liu L. Dudler T. Gelb M.H. J. Biol. Chem. 1996; 271: 23269-23276Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) The role of palmitoylation in localization and signaling functions of Gα, particularly the nonmyristoylated Gα (αs, αq, α12 classes), remains poorly understood. For the Gα that are both myristoylated and palmitoylated (αi class), mutations that abolish both modifications result in a cytoplasmic and soluble, rather than plasma membrane-bound, protein (4.Jones T.L. Simonds W.F. Merendino J.J. Brann M.R. Spiegel A.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 568-572Crossref PubMed Scopus (163) Google Scholar, 5.Mumby S.M. Heukeroth R.O. Gordon J.I. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 728-732Crossref PubMed Scopus (210) Google Scholar, 22.Hallak H. Brass L.F. Manning D.R. J. Biol. Chem. 1993; 269: 4571-4576Abstract Full Text PDF Google Scholar, 23.Galbiati F. Guzzi F. Magee A.I. Milligan G. Parenti M. Biochem. J. 1994; 303: 697-700Crossref PubMed Scopus (41) Google Scholar, 24.Wilson P.T. Bourne H.R. J. Biol. Chem. 1995; 270: 9667-9675Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Mutant Gα that remain myristoylated but are no longer palmitoylated are found in both membrane and cytosolic fractions and appear to be widely distributed among intracellular membranes and plasma membranes (15.Morales J. Fishburn C.S. Wilson P.T. Bourne H.R. Mol. Biol. Cell. 1998; 9: 1-14Crossref PubMed Scopus (80) Google Scholar). In contrast, results are less consistent from a number of studies of the nonmyristoylated Gα. Mutations of palmitoylated cysteines in αs, αq, α11, and α12 have revealed either little or no change in membrane association (10.Mumby S.M. Kleuss C. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2800-2804Crossref PubMed Scopus (219) Google Scholar,25.Degtyarev M.Y. Spiegel A.M. Jones T.L.Z. Biochemistry. 1993; 32: 8057-8061Crossref PubMed Scopus (85) Google Scholar, 26.Hepler J.R. Biddlecome G.H. Kleuss C. Camp L.A. Hofmann S.L. Ross E.M. Gilman A.G. J. Biol. Chem. 1996; 271: 496-504Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 27.Jones T.L. Gutkind J.S. Biochemistry. 1998; 37: 3196-3202Crossref PubMed Scopus (34) Google Scholar) or a shift of most of the Gα to soluble fractions (28.Wedegaertner P.B. Chu D.H. Wilson P.T. Levis M.J. Bourne H.R. J. Biol. Chem. 1993; 268: 25001-25008Abstract Full Text PDF PubMed Google Scholar, 29.Wise A. Parenti M. Milligan G. FEBS Lett. 1997; 407: 257-260Crossref PubMed Scopus (20) Google Scholar). In terms of signaling activity, studies with nonpalmitoylated mutants of αs, αq, α11, and α12 have consistently demonstrated a partial or complete loss of signaling in cultured cells (27.Jones T.L. Gutkind J.S. Biochemistry. 1998; 37: 3196-3202Crossref PubMed Scopus (34) Google Scholar, 28.Wedegaertner P.B. Chu D.H. Wilson P.T. Levis M.J. Bourne H.R. J. Biol. Chem. 1993; 268: 25001-25008Abstract Full Text PDF PubMed Google Scholar, 29.Wise A. Parenti M. Milligan G. FEBS Lett. 1997; 407: 257-260Crossref PubMed Scopus (20) Google Scholar). Although a lack of attached palmitate on Gα appears to result in a lack of signaling, the mechanism of this effect remains unclear. Defective signaling by cysteine mutants could be due to a failure to localize correctly at plasma membranes, an absence of palmitate-mediated protein-protein interactions, or the loss of important interactions mediated by the cysteine side chains. A goal of the studies presented here was to gain a better understanding of palmitoylation by analyzing its importance in localization and signaling by α13. α13, together with α12, comprise the α12 class of Gα, and these proteins regulate a variety of pathways involved in cell growth and differentiation (30.Dhanasekaran N. Tsim S.T. Dermott J.M. Onesime D. Oncogene. 1998; 17: 1383-1394Crossref PubMed Scopus (131) Google Scholar). Cellular activities regulated by α13 include oncogenic transformation of fibroblasts, Na+/H+ exchange, c-Jun N-terminal kinase and mitogen-activated protein kinase pathways, tyrosine phosphorylation, apoptosis, actin stress fiber formation, neurite retraction, focal adhesion assembly, and serum response element-mediated gene expression (30.Dhanasekaran N. Tsim S.T. Dermott J.M. Onesime D. Oncogene. 1998; 17: 1383-1394Crossref PubMed Scopus (131) Google Scholar). Some of these pathways require the small GTPase Rho, and indeed, α12 and α13 can stimulate the formation of active, GTP-bound RhoA in cultured cells (31.Kranenburg O. Poland M. van Horck F.P. Drechsel D. Hall A. Moolenaar W.H. Mol. Biol. Cell. 1999; 10: 1851-1857Crossref PubMed Scopus (270) Google Scholar). An important connection between α13 and Rho was revealed recently by the identification of the first direct effector for α13, a Rho-specific guanine-nucleotide exchange factor termed p115-RhoGEF (32.Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (671) Google Scholar). Interestingly, α12 was unable to activate p115-RhoGEF, indicating that this may be an α13-specific effector. Not only is p115-RhoGEF a direct effector for α13, p115-RhoGEF also can regulate α13, and to a lesser extent α12, by virtue of an RGS domain that stimulates the GTPase activity of α13 (33.Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (733) Google Scholar). In this report, we show that α13 is palmitoylated at cysteine 14 and/or cysteine 18 and demonstrate a palmitoylation-dependent plasma membrane anchoring of α13. Palmitoylation of α13 was also required for Rho-mediated serum response factor (SRF) activation and stress fiber formation. However, p115-RhoGEF interacted with both wild type and CS mutant of α13. Furthermore, we demonstrate that p115-RhoGEF can translocate from cytoplasm to plasma membrane in response to activated α13, but a nonpalmitoylated mutant of α13 fails to induce this translocation. These observations agree with the hypothesis that one of the major roles of palmitoylation is to tether the acylated protein(s), α13in this case, to plasma membranes and thus allow productive protein-protein interactions, either by increasing the acylated protein's concentration in the vicinity of interacting proteins or delivering other proteins (e.g. p115-RhoGEF) to important locations in the cell. The HA epitope (DVPDYA)-tagged pcDNAIIIHA G13wt and pcDNAIIIHA G13QL were kind gifts from J. S. Gutkind (34.Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar) and will hereafter be referred to as α13 and α13QL. α13QL, without an epitope tag, was provided by N. Dhanasekaran. The reporter plasmid that expresses the luciferase gene under the control of a mutant serum response element lacking a ternary complex factor binding site (SRF), termed pSRF-Luc, was purchased from Stratagene. The expression vector for Myc epitope-tagged C3-transferase, pEF-Myc-C3-transferase, was a kind gift from A. Hall (35.Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1197) Google Scholar). The plasmid encoding Myc epitope-tagged p115-RhoGEF, pEXV-Myc-p115-RhoGEF, was a gift from M. Hart (36.Hart M.J. Sharma S. elMasry N. Qiu R.G. McCabe P. Polakis P. Bollag G. J. Biol. Chem. 1996; 271: 25452-25458Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The plasmids carrying HA epitope-tagged activated RhoA cDNA (RhoV14) or the β-galactosidase gene (pCMV-β-gal) were obtained from P. Tsichlis (Kimmel Cancer Institute, Philadelphia). The Stratagene QuikChange Site-Directed Mutagenesis Kit was used to replace cysteines at positions 14 and 18 of α13. We mutated both cysteines separately and dually in the wild type and QL background of α13 by using two sets of forward and reverse primers carrying base changes in appropriate codons. The forward and the reverse primers used to mutate cysteine 14 to serine to create α13wtC14S or α13QLC14S, were 5′-ccgtgctgtccgtaagcttcccgggctgc-3′ and 5′-gcagcccgggaagcttacggacagcacgg-3′, respectively. Another set of primers, forward (5′-gtgtgcttcccgggctcagtactgacgaacggc-3′) and reverse (5′-gccgttcgtcagtactgagcccgggaagcacac-3′), were used to mutate cysteine 18 to serine to create α13wtC18S and α13QLC18S. The set of primers to mutate cysteine 18 to serine was utilized on C14S mutants to create double mutants, α13wtCCSS and α13QL CCSS. The correct DNA sequence was confirmed by DNA sequencing of the entire open reading frame (Kimmel Cancer Center Nucleic Acid Facility). HEK293, COS, and Rat-1 cells were propagated in Dulbecco's minimal essential medium containing 10% fetal bovine serum (unless otherwise mentioned) and Gentamicin. Unless otherwise noted, cells were plated in six-well plates at 7.0 × 105 cells per well and grown for 24 h prior to transfection. 1 μg of each expression plasmid was transfected into the cells using LipofectAMINE (Life Technology Inc.) or FuGene 6 (Roche Molecular Biochemicals). HEK293 cells were plated at 2 × 106 cells in 6-cm plates, grown for 24 h, and transfected with 3 μg of expression plasmid. 24 h after transfection, the cells were transferred to 10-cm plates and grown for another 24 h. Particulate (P) and soluble (S) fractions were prepared from cell lysates, and expressed proteins were analyzed by immunoblotting exactly as described previously (37.Evanko D.S. Thiyagarajan M.M. Wedegaertner P.B. J. Biol. Chem. 2000; 275: 1327-1336Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). We used the anti-HA antibody (12CA5) and the anti-Myc antibody (36.Hart M.J. Sharma S. elMasry N. Qiu R.G. McCabe P. Polakis P. Bollag G. J. Biol. Chem. 1996; 271: 25452-25458Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) to detect HA epitope-tagged proteins and Myc epitope-tagged proteins, respectively. 24 h after transfection in six-well plates, HEK293 cells were replated on glass coverslips and grown for 24 h. Cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS) for 15 min and permeabilized by incubation in blocking buffer (2.5% nonfat milk and 1% Triton X-100 in PBS) for 30 min. Cells were then incubated with appropriate monoclonal antibody (3 μg/ml), as mentioned in each figure legend, in blocking buffer for 1 h. The cells were washed with blocking buffer and incubated for 30 min in a 1:100 dilution of a fluorescently labeled secondary antibody. Donky anti-mouse antibodies labeled with Texas Red or fluorescein isothiocyanate (Jackson Immunoresearch, West Grove, PA) and goat anti-mouse antibodies labeled with Alexa488 (Molecular Probes, Inc., Eugene, OR) were used. The coverslips were washed with 1% Triton X-100 in PBS, rinsed in distilled water, and mounted on glass slides with 10 μl of Prolong Antifade reagent (Molecular Probes, Inc.). Microscopy was performed with an Olympus BX60 microscope equipped with a 60X/NA1.4 objective. Images were recorded with a Sony DKC-5000 digital camera and transferred to Adobe Photoshop for digital processing. Radiolabeling with [9,10-3H]palmitic acid (NEN Life Science Products) was performed exactly as described (37.Evanko D.S. Thiyagarajan M.M. Wedegaertner P.B. J. Biol. Chem. 2000; 275: 1327-1336Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). COS cells (4 × 105) were plated in 24-well plates and grown for 24 h. Cells were then switched to serum-free medium (with antibiotic) and transfected, using FuGene 6, with pCMV-β-gal (0.12 μg), pSRF-Luc (0.12 μg), and other plasmids as indicated. After 24 h of transfection, cells were carefully washed twice with cold PBS and were lysed by using reporter lysis buffer (Promega). 20 μl of each lysate was mixed at room temperature with 50 μl of luciferase substrate (Promega), and luciferase activities were determined by measuring luminescence intensity. Lysates were assayed for β-galactosidase activity by the colorimetric method and were used to normalize transfection efficiency. After allowing Rat-1 cells to attach to coverslips, the culture medium was changed to Dulbecco's minimal essential medium plus 0.1% fetal bovine serum. 24 h later, the culture medium was changed to Dulbecco's minimal essential medium without serum. 24 h after that, the cells on coverslips were transfected using FuGene 6 (Roche Molecular Biochemicals). Cells were then fixed with 3.7% formaldehyde 24 h after transfection. Coverslips were prepared for fluorescence microscopy, as described under "Immunofluorescence Localization," with the exception that a 1:40 dilution of Alexa594 conjugated to phalloidin (Molecular Probes) was included during the secondary antibody incubation to detect actin fibers HEK293 cells were transfected according to the earlier described protocol, with plasmid encoding Myc epitope-tagged p115-RhoGEF together with plasmids encoding HA epitope-tagged α13, α13QL, α13CCSS, and α13QL CCSS. After transfection, cells were washed twice with cold PBS and were lysed at 4 °C in 1 ml of buffer containing 25 mmHEPES, pH 7.5, 0.3 m NaCl, 1.5 mmMgCl2, 0.2 mm EDTA, 0.5 mmdithiothreitol, 1% Triton X-100, 0.5% 10 10 mm 20 mm 1 mm 1 mm 20 and 20 Lysates were at × for 10 min at 4 HA epitope-tagged α13 and its mutants were from the by for 1 h at 4 °C with anti-HA antibody. were with the of Inc., Lysates and the were analyzed by immunoblotting with appropriate monoclonal antibody as mentioned in each figure In we utilized anti-HA epitope (12CA5) and anti-Myc epitope (32.Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (671) Google Scholar) monoclonal of the amino acid sequence of α13 cysteine 14 and/or cysteine 18 as potential for palmitoylation. this we mutated the appropriate to replace cysteines 14 and 18 with serines, either and or mutated The double cysteine to serine mutant was also in α13QL a of α13 in which a at position is with a T. R. Bourne H.R. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). and and to transfected α13 from G protein α α13 and its mutants described here also a HA epitope at the N The of the HA not the ability of α13QL to activate signaling pathways (34.Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar) it palmitoylation of α13 of transcription by α13QL, but not by α13QL CCSS. pSRF-Luc (0.12 and pCMV-β-gal (0.12 plasmids were in COS cells with μg of μg of plasmid encoding activated μg of plasmid encoding activated α13 QL or μg of plasmid encoding activated RhoA In addition, included or μg of plasmid encoding 24 h after transfection, cells were and cell were assayed for luciferase activity and β-galactosidase activity as described under activity was by β-galactosidase activity in each are expressed as activity with to control cells and are the of results were obtained in other 14 and cysteine 18 are required for palmitoylation of α13. HEK293 cells were transfected with plasmid or with plasmids encoding HA epitope-tagged α13 α13QL or α13QL h after transfection, cells were incubated with acid in 6-cm were by using the antibody the HA proteins were to and were to and immunoblotting with antibody. a of a of a from cells transfected with plasmids as described in A and with acid of transfected HEK293 by with monoclonal demonstrated that α13 2 and α13QL is consistent with the previously described of palmitate into α13 after expression in cells M. B. C. E. G. FEBS Lett. 1994; PubMed Scopus Google Scholar). the 3 and and double cysteine to serine mutants 2 and of α13 failed to of that of α13 proteins were from the cell result that both cysteine 14 and cysteine 18 are required for palmitoylation of α13 and are the of attachment of A of an the lack of palmitoylation of the and double cysteine mutants 2 The failure of the cysteine to serine mutants, and to palmitate is to a with (28.Wedegaertner P.B. Chu D.H. Wilson P.T. Levis M.J. Bourne H.R. J. Biol. Chem. 1993; 268: 25001-25008Abstract Full Text PDF PubMed Google Scholar). α13, two N-terminal at positions and were required for palmitoylation of cysteine to serine mutants of very of the role of palmitoylation in proper membrane localization of α13, we assayed the subcellular localization of α13 and its cysteine to serine of transfected cells demonstrated the importance of palmitoylation for plasma membrane localization of α13. type α13 was at plasma membranes, as by at the cell However, both of the cysteine mutants, and and the double cysteine no plasma membrane but a cytoplasmic These results that palmitoylation by cysteine to serine mutations the ability of α13 to to plasma this of subcellular its mutants were assayed for their in and cytosolic fractions after cell HEK293 cells were lysed in a and cellular proteins were into (P) or soluble (S) fractions by that into the that α13 is tightly to cellular membranes in with results The mutants of α13 in the soluble fractions (S) 4 4 a of cellular that the of α13 and α13QL has to and for and α13QL respectively. into the by the α13 cysteine to serine mutants is consistent with their lack of palmitoylation and indicating a cytoplasmic localization it has that α13 can activation of the small G protein RhoA by G protein-coupled receptors (e.g. the acid A. R. G. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, of α13 can stimulate RhoA and signaling Thus, we two Rho-dependent signaling to the signaling ability of CS mutants of α13. signaling functions of α13 in a α13QL was with α13QL CCSS. we assayed the ability of α13QL or α13QL to activate gene transcription J. H. W. D. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). of gene transcription was in a transfection a reporter plasmid containing a serum response element that and the transcription of a luciferase of activated of the of the reporter luciferase