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z Inhibits Serum Response Factor-Dependent Transcription by Inhibiting Rho Signaling
Physiology Department, Tufts University School of Medicine, Boston, Massachusetts (P.D., K.D.M., D.T.); and Medical Research Council Laboratory for Molecular Cell Biology, Cancer Research UK Oncogene and Signal Transduction Group, University College London, London, United Kingdom (A.B.J., A.H.)
Received May 18, 2004; accepted August 23, 2004
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Abstract |
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12/13 or Gq signals induce activation of Rho GTPase, leading to serum response factor (SRF)-mediated gene transcription and actin cytoskeletal organization; however, less is known regarding how Rho pathway signals are down-regulated. Here we report that Gz signals inhibit serum response factor (SRF)-dependent transcription. Gz expression inhibits G12/13-, Gq-, and Rho guanine nucleotide exchange factor (GEF)-induced serum response element (SRE) reporter activation in human embryonic kidney 293T and PC-12 cells. Expression of Gz mutants with defective fatty acylation has no inhibitory effect. Expression of Gz, but not Gi, attenuates serum-induced SRE reporter activation, suggesting that Gz can down-regulate endogenous signals leading to SRF. Whereas Gz also blocks SRE reporter induction by the activated mutant RhoAL63, it does not affect G12- or Rho GEF-induced RhoA activation or RhoAL63-GTP binding in vivo. Moreover, Gz does not inhibit SRE reporter induction by an activated form of Rho kinase. Because Gz inhibits RhoAL63/A188-induced reporter activation, phosphorylation of RhoA on serine 188 does not seem to be involved; furthermore, RhoA subcellular localization was not affected. Use of pharmacologic inhibitors implies that Gz-induced reduction of SRE reporter activation occurs via a mechanism other than adenylate cyclase modulation. These findings suggest that Gz signals may attenuate Rho-induced stimulation of SRF-mediated transcription.
). At the molecular level, Rho controls a signaling pathway that regulates extracellular factor-induced actin filament assembly (Hall, 1998) and serum response element (SRE)-dependent gene transcription via the transcription factor serum response factor (SRF) (Hill et al., 1995). Defective Rho pathway signaling has been implicated in several diseases, including cancer and cardiovascular disease (Boettner and Van Aelst, 2002; Toksoz and Merdek, 2002); however, much remains to be understood regarding how this ubiquitous pathway is normally regulated.
Rho responses are induced by serum stimulation, and agonists for certain G protein-coupled receptors (GPCRs) such as lysophosphatidic acid induce actin polymerization and SRE transcriptional reporter activation via Rho (Kjoller and Hall, 1999; Seasholtz et al., 1999). These GPCR signals are transduced to Rho via heterotrimeric G12/13 and Gq family members through partially understood mechanisms that probably involve guanine nucleotide exchange factors (GEFs) (Seasholtz et al., 1999; Schmidt and Hall, 2002). GTPases such as Rho bind guanine nucleotides, and their activation state is determined by whether they bind GDP in the inactive state or GTP in the active state. Rho GEFs directly activate Rho by inducing rapid GDP/GTP exchange, resulting in activated GTP-bound Rho (Schmidt and Hall, 2002). Once activated, Rho signals are translated into downstream cellular responses via specific effectors (Bishop and Hall, 2000) such as Rho kinase (ROK).
Rho is required for actin filament assembly, which underlies phenotypic changes in cell shape, cell contraction, adhesion, and migration in most tissues. For example, Rho stimulates formation of actin stress fibers in fibroblasts (Ridley and Hall, 1992). In addition, Rho is required for extracellular factor-induced activation of SRF, which binds to the SRE found in many gene promoters, leading to transcription (Hill et al., 1995). In addition to regulating immediate-early genes such as c-fos and Egr-1, SRF regulates several skeletal and smooth muscle-specific genes, including - and 
-actin, which are required for differentiated tissue function, as well as ubiquitously expressed genes such as vinculin (Arsenian et al., 1998). The requirement for Rho in SRF function has led to the common use of SRE transcriptional reporter assays as a measure for Rho-dependent signals in vivo.
Although the requirement for Rho function in many cellular responses is now well established, there is also accumulating evidence that certain cellular responses require suppression of Rho function. For example, inhibition of Rho function has been reported to be required for processes such as dendritic outgrowth in melanocytes (Busca et al., 1998), development of neurite extensions (Jalink et al., 1994; Li et al., 2002), axon regeneration (Lehmann et al., 1999), vasopressin-mediated aquaporin-2 translocation (Klussmannet al., 2001), nitric oxide/cGMP/G kinase pathway-mediated transcriptional modulation (Gudi et al., 2002), and somatostatin-induced inhibition of cell migration (Buchan et al., 2002). Thus, Rho inhibition probably plays an integral role in particular stages of differentiation in certain cell types, particularly cellular responses. However, the pathways and mechanisms involved in Rho signal down-regulation are poorly understood.
Here we investigate the potential role of Gi family members in Rho signal down-regulation. Of the four G subunit families, G12/13, Gq, Gi, and Gs (Simon et al., 1991), Gi and Gs do not seem to be involved in the induction of Rho pathway signals. Results presented here suggest that signals by the Gi family member Gz inhibit Rho-mediated responses. Gz attenuates SRF-mediated transcription induced by G12/13/q signals, Rho GEFs, and serum. In addition, Gz blocks transcriptional activation by the constitutively active RhoAL63 mutant, although it seems to have no effect on transcriptional activation induced by the activated ROK downstream effector. Potential mechanisms for the observed inhibition of SRF-dependent transcription are investigated and discussed. Finally, the influence of Gz signals on actin cytoskeletal organization is evaluated.
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Materials and Methods |
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).
Plasmids. Wild-type Gz and activated mutant Gz Q205L, Gi1 Q204L, Gi2 Q205L, GoA Q205L, Gq Q209L, G12 Q231L, G13 Q226L, and Gs Q213L in pcDNA3.1 vector were obtained from the Guthrie cDNA Resource Center (Sayre, PA). Plasmids for pcDNA: GzG2A, pcDNA:GzG2A3A, RhoAL63/A188, SRE.L, TOPFLASH luciferase reporters, pEF C3 transferase, pGEX2T Rhotekin Rho binding domain (RBD), dominant-active ROK, and p115 Rho GEF were gifts. pSR:proto-Lbc Rho GEF is described in Sterpetti et al. (1999).
Antibodies and Reagents. Anti-RhoA, anti-G12 and anti-myc antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Protein kinase A inhibitor H-89 and adenylate cyclase inhibitor MDL-12 were obtained from Calbiochem (San Diego, CA).
Cell Transfection. Six-well dishes for transcriptional reporter assays or 100-mm dishes for RBD assays at 80% confluence were transfected with plasmid DNA for 5 h using LipofectAMINE Plus (Invitrogen) according to manufacturer's recommendation. Cells were serum-starved overnight and lysed the following day.
Immunoblotting. Cellular material was resolved by 10% SDS/polyacrylamide gel electrophoresis. Immunoblotting was carried out as described in Sterpetti et al. (1999).
Dual Luciferase Reporter Assay. SRE.L luciferase reporter plasmid, which encodes a mutant SRE that encodes functional SRF binding sites but eliminates the ternary complex factor (TCF) binding site (Hill et al., 1995), was used with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) as recommended. Inducible firefly luciferase results obtained were normalized to internal control Renilla reniformis luciferase values expressed from pTK-RL plasmid (Promega) after sequential measurement of the two luciferase activities. Each point was obtained in triplicate; experiments were repeated more than twice.
RBD Assay. In vivo GTP-Rho "pull-down" was carried out using GST-Rhotekin RBD fusion protein (Ren et al., 1999) as described previously in Dutt et al. (2002); experiments were repeated more than twice.
Subcellular Fractionation. HEK293T cell lysates were fractionated into S-100 soluble and P-100 particulate fractions as described in Sterpetti et al. (1999).
Microinjection. To prepare confluent quiescent, serum-starved Swiss 3T3 cells for microinjection, cells were seeded onto acid-washed coverslips at a density of 5 x 104 in DMEM containing 5% serum. After the cells became quiescent (approximately 7-10 days after seeding), they were serum-starved for 16 h in DMEM containing 2 µg/l NaHCO3. Eukaryotic expression vectors (0.1 µg/µl) together with biotin-dextran were injected into the nucleus of approximately 50 cells over a period of 15 min. Cells were returned to the incubator for 2 to 3 h for optimal expression, fixed in 4% paraformaldehyde for 10 min at room temperature, and stained for the epitope tag, injection marker, and actin as described in Nobes and Hall (1995). Fluorescence images were recorded on a charge-coupled device camera and processed using Openlab software.
Statistical Analysis. Data were analyzed using Student's t test; p values <0.05 were considered significant.
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Results |
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subunit family members on Rho pathway signals were tested by an in vivo transcriptional reporter assay based on the SRE.L luciferase reporter that encodes a mutant SRE that contains Rho-dependent SRF binding sites but eliminates the Ras-responsive TCF binding site (Hill et al., 1995). A dual luciferase reporter system was used that allows normalization of inducible luciferase activity readings against an internal Renilla luciferase standard provided by the cotransfected pTK-RL plasmid. As shown in Fig. 1A, expression of activated mutant Gs, Gi1, Gi2, Go, or Gz forms alone had minimal or no inductive effect on SRE luciferase reporter activity. Next we investigated potential inhibitory effects of Gi family members on inductive signals mediated by G12, G13, and Gq. As shown in Fig. 1A, coexpression of activated Gi1, Gi2, Go, or Gz separately by cotransfection of modest plasmid amounts (100 ng) each had a substantial reducing effect on G12/13 and Gq-induced reporter activity. Inhibition was observed in the ranges of 
40 to 60% by Gi1, 50 to 77% by Gi2, 37 to 54% by Go, and 70 to 85% by Gz. In contrast, coexpression of comparable amounts of GsQL had no inhibitory effect, suggesting that the effect is not caused by nonspecific G subunit coexpression. The inhibitory effects of Gi family members was not caused by cytotoxicity, because the internal R. reniformis luciferase control value levels were comparable with those of the inductive G subunit levels alone (not shown). Immunoblotting of total cell lysates shown in Fig. 1B revealed that levels of the stimulatory G subunit such as G12QL are not altered by coexpression of Gi family members, indicating that the inhibitory effect is unlikely to be caused by decreased G subunit expression. In addition, immunoblotting with anti-Glu-antibody showed that levels of expression of Glu-tagged activated Gi family members are comparable. Moreover, immunoblotting for endogenous RhoA showed that levels of total cellular RhoA were unchanged. In addition, we evaluated the effects of Gi2QL and GzQL expression on G12QL-induced activation of endogenous Rho by carrying out Rho pull-down experiments by affinity purification with Rhotekin RBD, which preferentially binds GTP-RhoA. As shown in Fig. 1C, Gi2QL coexpression had no effect on the levels of G12QL-induced GTP-RhoA, and GzQL coexpression with G12QL led to only a marginal reduction that was not statistically significant (p > 0.05).
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Next the effect of Gi family expression on Rho pathway components was tested. As expected and shown in Fig. 2A, expression of two different Rho GEFs, p115 and Lbc Rho GEF, led to SRE reporter induction via endogenous Rho. Coexpression of increasing levels of activated GzQL with Lbc or p115 Rho GEF caused inhibition of Rho GEF-induced SRE reporter activation, even at a 50-ng dosage of GzQL plasmid. As a control for the observed Gz effect, we used a Gz mutant, GzG2A, which has defective fatty acylation as a result of a point mutation that destroys the myristoylation site and thus abolishes Gz signaling ability (Morales et al., 1998). GzG2A coexpression had no inhibitory effect on Rho GEF-induced reporter activation, even at a 200-ng dosage (although wild-type Gz is inhibitory; see Fig. 4A). In contrast to its effect on G12/13/q-induced signals, expression of activated Gi2 had no blocking effect on Rho GEF-induced reporter activity, and the remaining Gi members also did not affect Rho-GEF signals (not shown). We next tested whether GzQL inhibits downstream Rho effectors such as ROK, which readily induces SRF-mediated responses (Sotiropoulos et al., 1999). Figure 2A shows that, in contrast to its effect on Rho GEFs, coexpression of increasing amounts of GzQL with activated ROK had minimal effect on ROK-induced SRE reporter activation. In addition, we evaluated the effect of GzQL expression on Lbc Rho GEF-induced activation of endogenous Rho by carrying out Rho pull-down experiments with Rhotekin RBD to preferentially purify the affinity of GTP-RhoA. As shown in Fig. 2B, coexpression of either Gi2QL or GzQL with Lbc Rho GEF did not have a noticeable effect on the levels of Lbc-induced GTP-RhoA in vivo.
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We next tested whether Gz can modulate endogenous Rho pathway signals induced by extracellular stimuli. As shown in Fig. 2C, stimulation of HEK293T cells by 15% serum treatment leads to SRE.L reporter activation, and this induction is Rho-dependent as determined by inhibition upon C3 transferase expression (Hill et al., 1995; not shown). It is interesting that expression of increasing amounts of GzQL (50-400 ng) in serum-stimulated cells led to a 40% reduction in SRE reporter activity, which was significant. In contrast, expression of Gi2QL did not inhibit serum-induced reporter activity. When the effect of GzQL expression on serum-induced GTP-RhoA levels was measured, a significant reduction was not observed (result not shown).
We next assessed whether the suppressive effect of Gz extends to other cell types by carrying out the same experiments in the PC-12 cell line, which is derived from adrenal pheochromocytoma tissue that normally expresses endogenous Gz (Ho and Wong, 2001). Although the transfection efficiency of PC-12 cells was lower than in HEK293T, Fig. 3A shows that GzQL expression had the same potent blocking effect on G12/13 and Lbc Rho GEF-induced SRE reporter activation in PC-12 cells as observed in HEK293T. Similar results were also obtained in 3T3 fibroblasts (not shown). To further investigate the Gz effect, we used a different transcriptional luciferase reporter in the form of the TOPFLASH luciferase reporter construct, which encodes binding sites for the LEF/TCF transcription factor induced by activated 
-catenin (Morin et al., 1997) but not by Rho (not shown). As shown in Fig. 3B, expression of the activated -catenin 
45 form (Morin et al., 1997) in HEK293T cells led to robust induction of TOPFLASH luciferase reporter that was unaffected by coexpression of GzQL at two different doses (100 and 400 ng).
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Because Gz inhibits signals to SRF/SRE by Rho GEFs but not by a downstream effector such as activated ROK, we next investigated potential effects on the activated GTPase-deficient RhoAL63 mutant. Figure 4A shows that coexpression of activated Gi1, Gi2, or Go with RhoAL63 had no inhibitory effect on SRE reporter activation. In contrast, coexpression of wild-type Gz or activated GzQL potently blocked RhoAL63-induced SRE reporter activation. As controls for the observed effect, we used two Gz mutants, GzG2A and GzG2A3CA, that have defective fatty acylation caused by point mutations that destroy the myristoylation and myristoylation plus palmitoylation sites, respectively, and thus abolish Gz signaling ability (Morales et al., 1998). As shown in Fig. 4, coexpression of Gz acylation mutants had no negative effect on RhoAL63-induced reporter activation. Immunoblotting of total cell lysates shown in Fig. 4B revealed that RhoAL63 protein levels were comparable in Gi, Go, or Gz coexpressing cells, indicating that the inhibitory effect of Gz was unlikely because of the decreased RhoAL63 expression. As expected for a constitutively GTPase-deficient mutant, GTP-Rho pull-down assay indicated that relative levels of GTP-RhoAL63 in vivo were not significantly reduced upon GzQL coexpression (not shown).
In light of these findings, we investigated potential mechanisms for the observed Gz-induced down-regulation. The downstream target of pertussis toxin-sensitive Gi family members is inhibition of adenylate cyclase activity (Wong et al., 1992), which leads to reduced protein kinase A (PKA) activity. Although not a strong candidate target of (PTX)-insensitive Gz, we nevertheless tested whether inhibition of adenylate cyclase or PKA activity by cell-permeable selective inhibitors leads to reduced Rho signals. Cells expressing RhoAL63, Lbc Rho GEF, GqQL, or G12QL were treated separately with the adenylate cyclase inhibitor MDL-12 or PKA inhibitor H-89. Figure 5A shows that neither of these pharmacologic agents significantly reduced SRE reporter activity induced by any of the stimuli used, although they blocked Gs-induced activation of a luciferase reporter encoding a cAMP response element binding protein site (not shown). Serine phosphorylation of RhoA at S188 blocks RhoA signaling function (Lang et al., 1996), and the RhoAL63/A188 mutant is resistant to this inhibitory modification. Figure 5B shows that coexpression of GzQL with RhoAL63/A188 led to a decrease in RhoAL63/A188-induced reporter activity, similar to its effect on RhoAL63/S188 signals.
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To determine whether Gz signals may lead to altered Rho subcellular localization, RhoAL63 was coexpressed with GzQL or GzG2GA in HEK293T, and cell lysates separated into cytosolic (soluble) and membrane-rich (pellet) fractions by high-speed fractionation. Figure 6 shows that, consistent with its activated state, a larger proportion of RhoAL63 localized to the pellet versus the soluble fraction when cotransfected with vector alone (Fig. 6, top). Coexpression of GzQL (Fig. 6, middle) or GzG2GA (Fig. 6, bottom) with RhoAL63 did not seem to alter the relative proportion of Rho in these fractions, as quantified by the graph. Additional experiments yielded the same outcome. Moreover, no changes in endogenous RhoA localization were observed under the same conditions (data not shown).
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We next evaluated whether Gz expression affects actin stress fiber formation, a cytoskeletal process that requires Rho function. For this purpose, quiescent Swiss 3T3 fibroblasts that contain few stress fibers were microinjected with a plasmid encoding an activated form of Net1 Rho GEF, Net1N (Alberts and Treisman 1998), along with either vector, GzQL or GzG2A. After 2 to 4 h, cells were fixed and stained for phalloidin to visualize actin. Figure 7B shows that Net1 Rho GEF microinjection led to increased stress fiber formation; moreover, this was not affected by coexpression of GzQL or GzG2A forms (Fig. 7, C and D), and these results are quantified by the graph in Fig. 7A. Similar results were obtained upon comicroinjection of RhoAL63 and GzQL (not shown).
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Discussion |
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i subunit family, Gz expression has a potent inhibitory effect on Rho-induced signals to SRF/SRE. Whereas Gz inhibits G12/13- and Gq-induced SRE reporter activation, it has no effect on G12-induced Rho activation as determined by measuring GTP-Rho levels in vivo (Fig. 1C). This indicates that Gz expression does not interfere with the signaling ability of G12 per se. Hence, it is unlikely that the observed inhibition was caused by Gz competition with other G subunits for regulators of G signaling, such as regulator of G protein signaling proteins (Ross and Wilkie 2000), activators of G protein signaling proteins, and/or / subunits (Blumer and Lanier 2003). Moreover, this finding implies that Gz-induced inhibition occurs downstream of G12/13/q. The Gi1 and Gi2 family members tested here also attenuated G12/13 and Gq-induced transcriptional signals to SRF, and, in this case, competition with other G subunits for G protein regulators cannot be ruled out; however, in contrast to Gz, Gi1 and Gi2 had no substantial effect on Rho GEF- or RhoAL63-induced reporter activation. The basis for this difference between Gi isotypes and Gz is not known at present, although such differences are consistent with other reports (Ho and Wong 2001) indicating a distinct function for Gz compared with other Gi family members. Gi is ubiquitously expressed, whereas Gz expression is more restricted and found in adrenal medulla, hypothalamus, retina, neural tissues, and platelets (Ho and Wong, 2001), although recent reports indicate that Gz may be expressed in a wider variety of tissues than previously thought (Hendry et al., 2000; Nagahama et al., 2002), and its precise role in signaling is poorly understood.
Our finding that Rho GEF-induced GTP-Rho levels in vivo is unaltered by Gz makes it unlikely that the observed block of Rho GEF-induced reporter activation by Gz was caused by inhibition of Rho GEF function, a notion compatible with the idea that the target of Gz action lies further downstream. The finding that Gz did not effectively block SRE reporter induction by activated ROK suggests that the Gz inhibitory effect may not extend to Rho effectors. However, although ROK induces SRF-mediated transcription under experimental conditions, it is not considered to be the main physiologic Rho effector that mediates SRF responses. The finding that Gz but not Gi2 expression partially blocks serum-induced SRE reporter activity suggests that Gz signals can attenuate endogenous signaling pathways to Rho and implies that the endogenous effectors of Gz are present in HEK293T. The use of Gz fatty acylation mutants indicates that the observed inhibition requires correct plasma membrane localization of Gz, a prerequisite for Gz signal transduction (Morales et al., 1998). Serum-induced signals leading to Rho-dependent responses are transduced by G12/13 and Gq (Seasholtz and Brown 1999); however, serum-induced GTP-RhoA formation was not affected by Gz signals (not shown). This further supports the notion that G signaling function per se is not affected by Gz and suggests that Gz targets a subsequent step on the signaling pathway. GPCRs, which may inhibit signaling to SRF/SRE via Gz, are not known at present; however, recently, stimulation of a GPCR (somatostatin GPCR) has been shown to inhibit Rho-dependent responses (Buchan et al., 2002) for the first time. This indicates the existence of GPCR-linked pathways that inhibit Rho and is consistent with our findings of Gz detailed here, although whether Gz is specifically involved in somatostatin-induced responses remains to be determined. The finding that Gz inhibits G12/13 and Rho GEF-induced SRE reporter in PC-12 neuronal cells that resemble Gz-expressing tissue suggests that the observed effect may also occur in Gz-rich tissues and is not restricted to HEK293T cells. The lack of effect of Gz signals on the LEF/TCF transcriptional reporter makes it unlikely that Gz signals block a common event required for transcriptional activation.
The observed inhibition of RhoAL63-induced SRE reporter activation by Gz coexpression is unlikely to be caused by modulation of a Rho regulator such as a Rho GEF or Rho GAP, because RhoAL63 activity is largely independent of these regulators. The lack of effect of GzQL on GTP-RhoAL63 levels supports the notion that Gz signals target the pathway at a point subsequent to Rho activation. The finding that wild-type Gz is nearly as active as GzQL in inhibiting RhoAL63 signals is notable although not unique, because transfected wild-type versions of G12/13 are also highly active in signaling to SRE luciferase reporter in HEK293T cells (Mao et al., 1998; Dutt et al., 2004). This probably reflects the ability of the sensitive luciferase reporter assay to detect some portion of the transfected wild-type G subunit that subsequently becomes activated in vivo.
The lack of effect of the adenylate cyclase and PKA inhibitors tested here on SRE reporter activity suggests that these signaling components (Wong et al., 1992) are not involved in the observed effect and is consistent with existing data showing that these components mainly transduce signals by Gi rather than Gz. Reports indicate the existence of Gi signaling pathways that involve effectors other than adenylate cyclase (Yang et al., 2002), and a number of alternate Gz effectors and mediators have been proposed (Ho and Wong, 2001). These include Rap1Gap (Meng et al., 1999), Rap1 GTPase (Woulfe et al., 2002), and G protein-regulated inducer of neurite outgrowth (Chen et al., 1999), and the potential involvement of these components in the modulation described here warrants further investigation.
A possible basis for the Gz-mediated effect is an effect on localization of a Rho signaling complex, and whereas altered RhoA subcellular localization in response to Gz was not detected here, it is conceivable that localization of other components of a RhoA signaling complex may be altered. In addition, Gz signals may lead to altered post-translational modification(s) of Rho. Phosphorylation of RhoA on Ser188 by cAMP or cGMP-dependent kinases inhibits Rho activity (Lang et al., 1996) by promoting Rho binding to cytosolic Rho guanine dissociation inhibitor (Ellerbroek et al., 2003). However, our finding that Gz inhibits RhoAL63/A188 signals shows that this modification is not responsible for the effect and, together with the RhoAL63/S188 result, implies a block downstream of Rho. Yet another possible basis for the observed results is inhibitory modification of other components of a Rho signaling complex.
Because we did not observe any effect on Rho GEF-induced stress fiber formation when we comicroinjected Gz in fibroblasts, this suggests that Gz interferes at a point in the SRF activation pathway that is independent of actin rearrangement; however, the possibility that the Gz expression level was insufficient to induce inhibition of stress fibers in this system cannot be ruled out. Moreover, additional assessment in cells other than fibroblasts, as well as on potentially more subtle effects on the actin cytoskeleton, is warranted. One potential target for the negative regulation of SRF/SRE by Gz is megakaryocytic acute leukemia, recently shown to be an SRF coactivator that responds to changes in levels of G-actin but has not been reported to regulate actin dynamics (Miralles et al., 2003). Another potential target is hCNK1, which is involved in Rho activation of SRF in an actin-independent manner (Jaffe et al., 2004). It is interesting that G kinase induces a similar effect in that it inhibits SRE/SRF transcription but does not seem to affect Rho-dependent cytoskeletal responses (Gudi et al., 2002). The Gz-induced transcriptional inhibition described here may play a role during certain stages of cell growth/differentiation that are accompanied by down-regulation of SRF-dependent genes. Thus, Gz signals may target a component(s) that selectively mediates SRF-dependent transcriptional responses, and elucidation of such signaling events would further contribute to our understanding of how this important transcriptional target is regulated.
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Acknowledgements |
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zG2A and GzG2AC3A, K. Kaibuchi for activated ROK, R. Pilz for RhoAL63/A188, M. Schwartz for pGEX:RBD, R. Treisman for SRE.L and pEFC3 transferase, B. Vogelstein for -catenin and TOPFLASH plasmids, and the Guthrie cDNA Resource Center (www.cdna.org) for G subunit plasmids. |
Footnotes |
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ABBREVIATIONS: SRE, serum response element; SRF, serum response factor; GPCR, G protein-coupled receptor; GEF, guanine nucleotide exchange factor; ROK, Rho kinase; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's medium; RBD, Rho binding domain; H-89, N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline; TCF, ternary complex factor; LEF/TCF, lymphoid enhancing factor/T cell factor; PKA, protein kinase A; RLU, relative luciferase units; MDL-12, cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-z-amine, HCL.
1 Current address: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892. 
Address correspondence to:Dr. Keith D. Merdek, Physiology Department, Tufts University School of Medicine, Boston, MA 02111. E-mail: keith.merdek{at}tufts.edu
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