Oncogene (2007) 26, 3122–3142 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW G Protein regulation of MAPK networks

ZG Goldsmith and DN Dhanasekaran

Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA, USA

G proteins provide signal-coupling mechanisms to hepta- the a-subunits has been used as a basis for the helical cell surface receptors and are criticallyinvolved classification of G proteins into Gs,Gi,Gq and G12 in the regulation of different mitogen-activated protein families in which the a-subunits that show more than kinase (MAPK) networks. The four classes of G proteins, 50% homology are grouped together (Simon et al., defined bythe G s,Gi,Gq and G12 families, regulate 1991). In G-protein-coupled receptor (GPCR)-mediated ERK1/2, JNK, p38MAPK, ERK5 and ERK6 modules by signaling pathways, ligand-activated receptors catalyse different mechanisms. The a- as well as bc-subunits are the exchange of the bound GDP to GTP in the a-subunit involved in the regulation of these MAPK modules in a following which the GTP-bound a-subunit disassociate context-specific manner. While the a- and bc-subunits from the receptor as well as the bg-subunit. The GTP- primarilyregulate the MAPK pathwaysvia their respec- bound a-subunit and the bg-subunit stimulate distinct tive effector-mediated signaling pathways, recent studies downstream effectors including enzymes, ion channels have unraveled several novel signaling intermediates and small GTPase, thus regulating multiple signaling including receptor tyrosine kinases and small GTPases pathways including those involved in the activation of through which these G-protein subunits positivelyas well mitogen-activated protein kinase (MAPK) modules as negativelyregulate specific MAPK modules. Multiple (Johnson and Dhanasekaran, 1989; Hepler and Gilman, mechanisms together with specific scaffold proteins that 1992; Dhanasekaran et al., 1995; Dhanasekaran and can link G-protein-coupled receptors or G proteins to Prasad, 1998; Rozengurt, 1998). distinct MAPK modules contribute to the context-specific MAPK modules regulate many critical signaling and spatio-temporal regulation of mitogen-activated pathways including cell proliferation, differentiation protein signaling networks byG proteins. and apoptosis (Fanger et al., 1997; Minden and Karin, Oncogene (2007) 26, 3122–3142. doi:10.1038/sj.onc.1210407 1997; Dhanasekaran and Premkumar Reddy, 1998; Gutkind, 1998; Garrington and Johnson, 1999; Davis, Keywords: GPCR; signal transduction; G protein; 2000; Chang and Karin, 2001). Each of these modules MAPK; oncogene; cancer consists of at least three distinct kinases, namely an upstream mitogen-activated protein (MAP) kinase kinase kinase (MAP3K), a MAP kinase kinase (MAP2K) and a downstream MAP kinase (MAPK). In a typical signaling pathway, growth factor stimulated Introduction small GTPases such as Ras, CDC42, and Rac activate an upstream MAP kinase kinase kinase kinase Heterotrimericguanine nucleotide-binding proteins, (MAP4K), following which the MAP kinase cascades or G proteins, consisting of a-, b-andg-subunits, proceed through the sequential phosphorylation of transduce signals from heptahelical cell surface receptors constituent MAP3K, MAP2K and MAPK (Minden to intracellular effectors (Simon et al., 1991; Hepler and Karin, 1997; Dhanasekaran and Premkumar and Gilman, 1992; Conklin and Bourne, 1993). The Reddy, 1998; Davis, 2000; Chang and Karin, 2001). a-subunit is a 37–42 kDa protein, which contains Activated MAP kinases translocate from the cytosol to the guanine nucleotide-binding pocket along with the the nucleus where they regulate the activities of various intrinsicGTPase activity.The 35-kDa b-subunit and the transcription factors through phosphorylation (Khokh- 8–11-kDa g-subunit are tightly associated with each latchev et al., 1998; Brunet et al., 1999). GPCRs, other and are often referred to as a single entity, the primarily through their cognate G proteins, regulate bg-subunit (Simon et al., 1991; Hepler and Gilman, these three-tier MAPK phospho-relay systems to 1992; Conklin and Bourne, 1993). To date, 17 different modulate the expression of specific primary as well as a-subunits, five b-subunits and 12 g-subunits have secondary response genes involved in cell proliferation, been identified. However, the amino acid identity of differentiation and apoptosis (Gutkind, 2000; Kranen- burg and Moolenaar, 2001; Werry et al., 2005). G proteins have been shown to regulate diverse transcrip- Correspondence: Dr DN Dhanasekaran, Fels Institute for Cancer Research and Molecular Biology, Temple University School of tion factors such as AP-1 (Araki et al., 1997), NF-kB, Medicine, Philadelphia, PA 19140, USA. CRE, SRE (Takeuchi and Fukunaga, 2003), ATF1 E-mail: [email protected] (Ghosh et al., 2002), STAT3 (Sellers et al., 1999), ELK1 G Proteins and MAPKs ZG Goldsmith and DN Dhanasekaran 3123 (Todisco et al., 1997), Egr-1 (Gerasimovskaya et al., studies focused on defining the oncogenic pathways 2002), HIF-1a (Sodhi et al., 2000), MEF2 (Fukuhara regulated by the gsp oncogene that encodes the et al., 2000a, b) via ERK-, JNK-, p38MAPKs- or ERK5 constitutively activated mutants of Gas (Landis et al., modules. However, only recently the dynamic and 1989; Lyons et al., 1990). Functional characterization of multilayered mechanisms by which G proteins regulate these activating mutations indicated that the ectopic these MAPK networks have begun to be unraveled. This expression of Gas could stimulate ERK1/2 in different review is focused on analysing the mechanisms by which cell types (Faure et al., 1994). Using mutant S49 G proteins regulate different MAPK networks in a lymphoma cell lines that lack Gas or PKA, it was context-specific manner. further established that Gas and PKA are required to couple b2-AR signaling to ERK1/2 module (Wan and Huang, 1998). Analysis of such Gas-mediated stimula- tion of ERK1/2 indicated that it was mediated by a Regulation of MAPKs byG s pathway involving Rap-1 and its activation of B-Raf, one of the isoforms of Raf (Vossler et al., 1997; Wan G , defined by the a-subunit Ga , transduces signals s s and Huang, 1998; Schmitt and Stork, 2000; Weissman from G -coupled heptahelical receptors to adenylyl s et al., 2002; Dumaz and Marais, 2005; Houslay, 2006; cyclase that converts ATP to cAMP and subsequently Wang et al., 2006b). One of the mechanisms through to cAMP-mediated activation of protein kinase A which Ga activates Rap-1 involves cAMP-mediated (PKA) (Gilman, 1987; Hepler and Gilman, 1992). s direct activation of EPAC (exchange protein directly Consequently, as depicted in Figure 1, cAMP as well activated by cAMP), a guanine nucleotide exchange as cAMP-activated PKA have been shown to play a factor (GEF) specific for Rap-1 (de Rooij et al., 1998; major role in Ga -mediated stimulation as well as inhi- s Quilliam et al., 2002; Bos, 2006). In rat kidney cortical bition of MAPK modules such as those of ERK1/2, collecting duct cells, it has been shown that the ERK5 and p38MAPK (Cook and McCormick, 1993; stimulation of Ga and subsequent generation of cAMP Graves et al., 1993; Wu et al., 1993; Wan and s leads to the activation of EPAC, which stimulates Huang, 1998; Zheng et al., 2000; Laroche-Joubert GDP–GTP exchange in Rap-1. The GTP-bound Rap-1, et al., 2002; Stork and Schmitt, 2002; Dumaz and thus stimulated, activates B-Raf and the downstream Marais, 2005; Houslay, 2006; Pearson et al., 2006). ERK1/2 modules (Laroche-Joubert et al., 2002). The observation that the introduction of antibodies to Epac-1, Gas stimulation of ERK1/2 Rap-1 and B-Raf into these cells inhibited Gs-mediated The initial evidence that Gas is involved in the activation of ERK1/2 module established the linear regulation of ERK1/2 signaling module consisting pathway leading from cAMP to the activation of of a MAP3K–MAP2K–MAPK module defined by ERK1/2 module involving Epac-1, Rap-1 and B-Raf Raf, MEK1/2 and ERK1/2, respectively, came from (Laroche-Joubert et al., 2002).

Adenylyl
Cylclase P s P cAMP

Ras EPAC PKA Src C-Raf C3G MEK-1/2 ERK-1/2 Rap1 MEKK2/3 MAP3K MEK5 MKK3/6 ERK5 B-Raf p38MAPK MEK-1/2 ERK-1/2

Figure 1 Gs regulation of ERK1/2. Gas stimulates the B-Raf-mediated activation of ERKs via Rap-1 or Ras and inhibits C-Raf- mediated activation of ERKs by phosphorylating C-Raf through PKA. Gas has also been shown to stimulate p38MAPK and inhibit ERK-5 modules via PKA-dependent mechanism. The dashed lines indicate the pathways that have not been fully resolved.

Oncogene G Proteins and MAPKs ZG Goldsmith and DN Dhanasekaran 3124 Although further studies confirmed the role of Rap-1 ERK pathway involves PKA-mediated phosphorylation in Gas-stimulated activation of ERK1/2 module, they of a specific isoform of Raf, known as Raf-1 or C-Raf also indicated that Gas stimulation of ERK1/2 module (Wu et al., 1993; Dhillon et al., 2002). C-Raf contains via Rap-1 involves a Rap-1 GEF other than EPAC in three PKA-target Serines, namely Ser 43, 259 and 621 many cell types (Stork and Schmitt, 2002). The cAMP (Morrison et al., 1993), all of which can be phosphory- analog, 8-(4-chloro-phenylthio)-20-O-methyladenosine- lated in vitro by PKA (Wu et al., 1993; Hafner et al., 30,50-cyclic monophosphate (8CPT-2Me-cAMP) that 1994; Mischak et al., 1996). Mutational analyses of can activate EPAC, but not PKA, proved useful in C-Raf indicated that the Gas-mediated cAMP–PKA identifying the alternate pathway through which Gas signaling axis inhibits C-Raf through the phosphoryla- activated ERK1/2 via Rap-1 (Enserink et al., 2002). The tion of Ser-259 (Dhillon et al., 2002). The PKA observations that 8CPT-2Me-cAMP stimulated EPAC phosphorylation of Ser-259 of C-Raf attenuates the but failed to activate Rap-1-dependent activation of phosphorylation of Ser-338, which is involved in the ERK suggested that EPAC might not be involved in activation of C-Raf (Dhillon et al., 2002), thus providing Rap-1-mediated activation of ERK1/2 module in all a molecular basis for Gas-mediated inhibition of C-Raf– the cells (Enserink et al., 2002). Further studies have MEK1/3–ERK1/2 module. In addition, it has been indicated that in many cell types, Gas-mediated activa- shown that Gas-cAMP–PKA-mediated activation of tion of ERK1/2 signaling via Rap-1 involves novel Rap- Rap-1 is also involved in the inhibition of C-Raf 1-GEF known as Crk SH3 domain-binding guanine containing ERK module in fibroblast cell lines such as nucleotide-releasing factor, or C3G (Tanaka et al., 1994; NIH3T3 cells (Schmitt and Stork, 2001, 2002b). This Gotoh et al., 1995). It should be noted here that unlike has been primarily attributed to the close sequence and the direct activation of EPAC by cAMP (de Rooij et al., structural similarities of the effector domain of Rap-1 1998; Quilliam et al., 2002; Bos, 2006), the activation with that of Ras, through which Rap-1 binds and of C3G requires the PKA-mediated phosphorylation sequesters C-Raf from being activated by Ras (Bos, of Ser-17 of Src(Schmitt and Stork, 2002a; Obara 1998; Schmitt and Stork, 2001, 2002b). et al., 2004; Weissman et al., 2002; Wang et al., In summary, it can be surmised that Gas stimulates 2006b), subsequent Src-mediated activation of Cbl, B-Raf–MEK–ERK1/2 module via Rap-1 pathway in Cbl-mediated recruitment of Crk–C3G complex and cells that express B-Ras. However, Gas stimulates or Crk-SH3 domain-mediated recruitment as well as inhibits C-Raf–MEK1/2–ERK1/2 modules dependent activation of C3G (Ichiba et al., 1999; Schmitt and on the cell type in which C-Raf is expressed. Whereas Stork, 2000, 2002a). Results from these studies establish Gas-mediated stimulation of C-Raf–MEK1/2–ERK1/2 a paradigm in which Gas stimulates ERK1/2 via a module involves PKA-dependent or -independent pathway involving cAMP–PKA–Src-mediated activa- activation of Ras, Gas-mediated inhibition involves tion of C3G, C3G stimulation of Rap-1 and, finally, PKA-mediated phosphorylation or Rap-1-mediated Rap-1-mediated activation of B-Raf-MEK–ERK sequestration of C-Raf. In light of recent findings that module (Schmitt and Stork, 2000; Weissman et al., adenylyl cyclase as well as PKA are functionally and/or 2002; Obara et al., 2004; Wang et al., 2006b). physically compartmentalized through their interactions Finally, it has also been noted that Gas-cAMP- with proteins such as ERKs (Gros et al., 2006) or mediated activation of Ras is involved in the stimulation AKAPs (Dumaz and Marais, 2005; Houslay, 2006), it is of ERK1/2 modules, as demonstrated in cortical quite possible that the Gas-mediated differential regula- neurons (Ambrosini et al., 2000), thyrocytes (Tsygankova tion of ERK modules by Gas involves different pools of et al., 2000), melanocytes (Busca et al., 2000) and adenylyl cyclase and/or PKA in a single cell. melanoma cell lines (Busca et al., 2000; Amsen et al., 2006), in addition to COS-7 as well as HEK293 cells (Norum et al., 2003, 2005). In these cells, Gas stimulates Gas regulation of p38MAPK and ERK5 Ras and the subsequent activation of ERK1/2 modules In addition to its role in the regulation of ERK1/2 via PKA-dependent or -independent mechanism in a cell- modules, Gas is involved in the regulation p38MAPK as type-specific manner. Thus, Gas-mediated PKA-depen- well as ERK5 modules through the cAMP–PKA dent stimulation of Ras signaling to ERK1/2 module has signaling axis. In cardiomyocytes, it has been demon- been shown to involve a PKA-dependent Ras-GEF strated that the b2-AR stimulates the activity of p38 known as Ras-GRF1 in COS-7 and HEK293 cells MAPK involving the classical Gas-cAMP–PKA me- (Norum et al., 2005, 2007), whereas PKA-independent chanism (Zheng et al., 2000). Although the mechanism signaling involves a cAMP-responsive but PKA-indepen- through which Gas couples to the p38MAPK module is dent Ras-GEF known as CNrasGEF in melanoma cell far from clear, the findings that Rap-1 can stimulate lines (Amsen et al., 2006). p38MAPK in diverse cell types (Ahn et al., 2005, 2006) point to the possible role of Rap-1 in this pathway. However, such a role for Rap-1 in Gas-mediated Gas inhibition of ERK1/2 activation of p38MAPK remains to be established. Gas-mediated cAMP–PKA signaling pathway has also In the case of ERK5, it has been established that been shown to potently inhibit ERK pathways (Cook the cAMP–PKA pathway is involved in inhibition of and McCormick, 1993; Graves et al., 1993; Wu et al., the ERK5 module consisting of MEKK2, MEK5 and 1993). A primary mechanism by which Gas inhibits the ERK5 (Pearson et al., 2006). Interestingly, it has been

Oncogene G Proteins and MAPKs ZG Goldsmith and DN Dhanasekaran 3125

PI3K Adenylyl α i Cylclase P ββγ γ P cAMP Rap1 PLC β PKA TK ? GAPII Ras Ca++-CaM Pyk2 Rho/ CDC42 Rap1 Src Shc C-Raf

SOS ? MEK-1/2 ? ERK-1/2

JNK Ras Rac

C-Raf MEK-1/2 MAP3K ERK-1/2 MKK4/7 JNK

Figure 2 Gi regulation of MAPK network. Gi stimulates the activities of ERK, JNK and p38MAPK through a- as well as bg-subunits-dependent mechanisms as depicted. See text for details.

observed that pretreatment of cells with cAMP inhibited that Gai2 can activate the ERK module through a receptor tyrosine kinase (RTK)-mediated activation diverse set of mechanisms involving either direct or of ERK5 in HeLa, C2C12 and NIH3T3 cells. Analysis indirect activation of Ras (Pace et al., 1995; Edamatsu of the underlying mechanism has indicated that et al., 1998). In addition to its activation of ERK PKA phosphorylates MEKK2 at Ser 153, the phos- modules through these mechanisms, Gai also appears to phorylation of which inhibits its coupling to transmit signals to ERK as well as other MAPK upstream activators, presumably the candidate modules via its erstwhile associated bg-subunit (Figure 2; MAP4Ks. Thus, Gas-cAMP–PKA-mediated phosphor- Crespo et al., 1994; Faure et al., 1994; Koch et al., 1994; ylation of MEKK2 potentially leads to the overall Pace et al., 1995). inhibition of signaling by MEKK2–MEK5–ERK5 module (Pearson et al., 2006). Gai stimulation of ERK1/2 Gai-dependent activation of ERK primarily involves the suppression of two inhibitory pathways. One mechan- Regulation of MAPKs byG i ism involves its inhibitory effect on adenylyl cyclase, whereas the other involves its inhibitory effect on Rap-1 The Gi family of G proteins is defined by the a-subunits via Rap-1-GAP protein. The well-characterized signal Gai1,Gai2,Gai3,Gaz,GaoA and GaoB. The identifica- transduction role of Gai is to couple specific receptors to tion of the activated mutants of Gai2 as the gip2 the inhibition of adenylyl cyclase (Johnson and Dhana- oncogene in pituitary adenomas as well as tumors of the sekaran, 1989; Tang and Gilman, 1992). Consistent with ovary and the adrenal cortex (Lyons et al., 1990) this prototypicsignaling role of G ai, expression of gip2, indicated the mitogenic activity of Gai. Studies focused the activated mutant of Gai, resulted in a decrease in the on defining the molecular basis for such mitogenic accumulation of cAMP in cultured cells (Lowndes et al., activity identified the role of Gai in the regulation of the 1991; Wong et al., 1991). Such a Gai-mediated decrease ERK module (L’Allemain et al., 1991; Pouyssegur and in cAMP levels and subsequent decrease in PKA activity Seuwen, 1992b). Expression of the GTPase deficient, relieves the inhibitory effect of PKA on C-Raf, thus constitutively activated mutant of Gai2 resulted in the potentiating Ras–C-Raf signaling to ERK module oncogenic transformation of Rat1 fibroblasts (Pace (Radhika and Dhanasekaran, 2001). The observation et al., 1991; Gupta et al., 1992b). Functional characteri- that the expression of Gai2 slightly enhances the zation of the transformed phenotype indicated that activation of C-Raf (Pace et al., 1995) is consistent with expression of the activated Gai2 was associated this view. with constitutive activation of p44-ERK activity However, Gai has also been shown to activate (Gupta et al., 1992a). Subsequent studies have shown the ERK pathway via an alternate Ras-dependent

Oncogene G Proteins and MAPKs ZG Goldsmith and DN Dhanasekaran 3126 mechanism (Pace et al., 1995; Mochizuki et al., 1999). the bg-subunit is involved in activation of ERK by a Using a strategy wherein the expression of a pertussis number of Gi-coupled receptors including m2-muscarci- toxin-resistant mutant of Gai2 in cells treated with PTX nic, a2-adrenergic, D2 dopamine, A1 adenosine and could be used to monitor the effect of A1-adenosine LPA receptors (Crespo et al., 1994; Koch et al., 1994). receptors specifically coupled to the transfected PTX- Consistent with these observations, expression of bg- resistant Gai2; it was demonstrated that the expression subunit has been shown to increase the basal activity of of Gai2 and subsequent activation by A1 adenosine ERK in COS-7 cell (Faure et al., 1994). Further analyses receptor led to the stimulation of ERK activity (Pace using a dominant negative mutant of Ras have indicated et al., 1995). In addition, the coexpression of dominant that the bg-mediated activation of ERK1/2 module negative mutant of Ras inhibited Gai2-mediated activa- involves Ras (Crespo et al., 1994; Faure et al., 1994; tion of MEK and the downstream ERK, indicating a Koch et al., 1994). Although bg-subunit-mediated role for Ras in this pathway (Pace et al., 1995). activation of ERK module has been shown to involve However, an intriguing observation was that there was Ras (Crespo et al., 1994; Koch et al., 1994), the no increase in the GTP-bound active form of Ras upon mechanism through which the bg-subunit couples to the stimulation of Gai2 by A1-adenosine receptors. Ras appears to be cell-type specific. However, the Thus, although these studies indicated that the activa- consensus is that the activation of Ras by bg-subunit tion of ERK by Gai2 required functional Ras, the involves a tyrosine kinase, the identity of which is molecular basis for such requirement with little or no context specific. The immediate step involved in bg- modulation of the activation Ras was not clear. mediated activation of ERK involves phospholipase- However, the later observation that Gai2 can interact C-b (PLC) and/or phosphoinositide-3-kinase (PI3K), with Rap-1-GAP provided a molecular basis for a both of which can be activated by the bg-subunit mechanism in which Gai2 can activate ERK through a (Camps et al., 1992; Katz et al., 1992; Stephens et al., Ras-sensitive pathway without directly activating Ras 1994; Thomason et al., 1994). Transfection studies using (Mochizuki et al., 1999). Rap-1 was initially identified as COS-7 or HEK293 cells have established a model in a Ras-like GTPase that can act as a tumor suppressor which Gi-coupled a2A-adrenergicreceptors stimulate by attenuating Ras-mediated cellular transformation ERK1/2 modules via the released bg-subunits (Lev (Pizon et al., 1988; Kitayama et al., 1989). Rap-1-mediated et al., 1995; Dikic et al., 1996; Della Rocca et al., 1997). suppression of transformation is associated with the In this model, bg-subunit released from Gi-heterotrimer ability of activated Rap-1 to attenuate Ras-dependent stimulates PLCb leading to phosphatidylinositol ERK activation by EGF as well as LPA (Cook et al., trisphosphate (IP3)-mediated increase in intracellular 1993). As described earlier, activated Rap-1 through Ca2 þ and Ca2 þ –calmodulin-mediated activation of Pyk2 its Ras-homologous effector domain sequesters C-Raf kinase. The Pyk2 kinase thus stimulated, activates Src, (Bos, 1998; Schmitt and Stork, 2001, 2002b), thus which in turn stimulates Shc adaptor protein leading to disrupting Ras-mediated signaling to the C-Raf– the activation of Ras via the Ras-GEF, mSOS MEK–ERK-signaling module. Gai2 attenuates the (Lev et al., 1995; Dikic et al., 1996; Luttrell et al., Ras-antagonistic inhibitory activity of Rap-1 by recruit- 1996; Della Rocca et al., 1997). ing a specific Rap-1 GTPase activating protein II In some other cellular contexts, it has been observed, (Rap1GAPII), which can downregulate Rap-1 activity bg-subunit transmits signals through an Src- and Shc- (Mochizuki et al., 1999). Thus, mutational or receptor- independent mechanism involving the phosphorylation mediated activation of Gai2 facilitates the translocation of dynamin II and its association with Grb2 (Kranen- of Rap1GAPII to the plasma membrane where Rap1- burg et al., 1997, 1999a, b). In Rat1a and COS-7 cells, it GAPII accelerates the GTPase activity of Rap-1, there- has been demonstrated that LPA- or thrombin receptor by rendering it inactive. Subsequent release of stimulated release of bg-subunit activates ERK1/2 sequestered C-Raf leads to Ras–C-Raf reassociation module via a pathway involving PI3K and PI3K- and the activation of the downstream ERK1/2 module mediated activation of a tyrosine kinase that promotes (Mochizuki et al., 1999). It is significant to note here dynamin II–Grb2 complex formation with the resultant that the pathway deduced from this signaling paradigm activation of Ras and the ERK1/2 module (Kranenburg supports a view that Gai2 stimulates Ras-dependent et al., 1997, 1999a, b). Since dynamin can link Ras-GEF ERK signaling without directly involving the activation SOS to Ras (Wunderlich et al., 1999), it is possible that of Ras by inhibiting the activity of Rap-1. the dynamin facilitates bg-mediated activation of Ras in this complex. As dynamin has been shown to play a critical role in vesicular endocytosis (Hinshaw and Gbgi stimulation of ERK1/2 Schmid, 1995; Praefcke and McMahon, 2004), and Gi has also been shown to stimulate the ERK1/2 vesicular endocytosis has been shown to be required for signaling module by mechanisms that involve the H-Ras-mediated activation of ERK1/2 module (Roy disassociated bg-subunits (Crespo et al., 1994; Koch et al., 2002), it is likely that dynamin II-mediated et al., 1994). Expressing the bg-subunit interacting endocytosis plays a critical role in bg-mediated activa- COOH domain of the b-adrenergicreceptor kinase tion of ERK1/2. As seen in the case of b-arrestin- (bARK-CT) or Gat, both of which can function as mediated activation of ERK1/2 in endocytic vesicles antagonists of bg signaling by virtue of their ability to (Lefkowitz and Shenoy, 2005), it is possible that sequester endogenous bg, it has been demonstrated that bg-mediated ERK1/2 signaling is not involved in the

Oncogene G Proteins and MAPKs ZG Goldsmith and DN Dhanasekaran 3127 transmission of signals to the nucleus. However, such a et al., 1999, 2000), presumably involving tyrosine kinase differential role for bg-dynamin-mediated activation of responsive-specific GEFs (Shou et al., 1995; van Biesen ERK1/2 modules remains to be clarified. et al., 1995; Miyamoto et al., 2003). Similar to the activation of the ERK module by Gbgi-subunit, the activation of JNK by Gbgi appears to require a PI3K Gi stimulation of JNK pathway activity in certain cellular contexts, such as the activa- In addition to the activation of ERKs, Gai as well as the tion of Gi-coupled m-opioid receptor (Kam et al., 2004). bg-associated with Gai has been shown to activate the JNK module (Coso et al., 1995, 1996; Edamatsu et al., 1998; Yamauchi et al., 2000). Stimulation of ectopically Regulation of MAPKs byG q expressed m2-muscharinic receptors that couple to Gi- family of G proteins have been shown to activate JNK The G family of G proteins is defined by the via bg-subunits in COS-7 cells (Coso et al., 1995, 1996). q Similar analysis using mastoparan, which mimics GPCR ubiquitously expressed Gaq,Ga11 and hematopoetic cell-specific Ga , and Ga subunits (Simon et al., activation by directly stimulating G proteins (Higashi- 14 15/16 1991; Hubbard and Hepler, 2006). All of the family jima et al., 1990), indicated that JNK could be activated members have been shown to activate ERK1/2, JNK by both the a- and the bg-subunits of G in HEK293 cells i and p38MAPK modules. The a-subunits of G family (Yamauchi et al., 2000). The use of bg-quenching q of G proteins transduce signals from their cognate bARK-CT established that the mastoparan activation receptors to specific cellular responses via the activation of JNK involved both Ga and Gbg in these cells i i of the effector PLCb (Johnson and Dhanasekaran, (Yamauchi et al., 2000). Consistent with this observa- 1989; Simon et al., 1991). Activated PLCb hydrolyses tion, transient expression of the constitutively activated phosphatidylinositol 4,5-bisphosphate (PIP2) to pro- mutants of Gai1,Gai2 and Gai3 in HEK293 cells increased JNK activity. Interestingly, similar constitu- duce inositol triphosphate (IP3) and diacylglycerol (DAG), both of which can activate protein kinase C tively activated mutants of Ga and Ga , which belong o z (PKC), either directly or indirectly via the release of to Gi family, failed to stimulate such JNK activation 2 þ (Yamauchi et al., 2000). Thus, the activation of JNK internally stored Ca . In addition to these a-subunit- mediated pathways, the bg-subunit released from G can appears to be restricted to some G isoforms but not to q i also activate PLCb (Camps et al., 1992: Katz et al., other G family members such as Ga or Ga . The i o z 1992) and transmit signals through both PLC–DAG– expression of dominant negative Rho and Cdc42, but PKC- and PLC–IP3-Ca2 þ -mediated signaling pathways not dominant negative Rac, inhibited Ga -mediated i (Figure 3). activation of JNK, suggesting a pathway involving Rho and CDC42 in the activation of JNK by Gai (Yamauchi et al., 2000). Interestingly, Gai-mediated activation of Gq stimulation of ERK1/2 JNK was not inhibited by the dominant negative Gaq can stimulate ERK1/2 module via PLC–DAG– mutants of MKK4 and MKK7, the upstream MAP2Ks PKC as well as PLC–IP3-Ca2 þ signaling mechanisms. that are typically involved in the activation of JNKs Gaq-activated PKC can stimulate ERK1/2 module by (Yamauchi et al., 2000). Thus, although, Rho and directly phosphorylating and stimulating C-Raf (Kolch Cdc42 have been shown to stimulate JNK via MKK4 in et al., 1993; Ueda et al., 1996; Schonwasser et al., 1998). 2 þ HEK293 T cells (Teramoto et al., 1996), Gai-mediated An alternate mechanism involving Ca –calmodulin- activation of JNK proceeds through a novel pathway mediated activation of Pyk2 leading to the activation of involving Rho and/or CDCD42 and a hitherto unchar- Ras and subsequently ERK1/2 module can also be acterized tyrosine kinase via an MKK4/7-independent envisaged (Lev et al., 1995; Dikic et al., 1996; Della mechanism (Yamauchi et al., 2000). In contrast, the Rocca et al., 1997). However, whether Gaq/11 activates 2 þ pathways involving bg-subunit dissociated from Gai ERK1/2 via the PKC–Raf signaling axis or IP3-Ca – have been demonstrated to involve Ras and Rac(Coso calmodulin–Pyk2–Src–Ras pathway appears to be cell et al., 1996). In addition, bg-mediated stimulation of type dependent. It has been shown that m1-muscarinic JNK involves both MKK4 and MKK7, as bg-activated receptor-mediated activation of Gaq stimulates the JNK can be inhibited by the dominant negative mutants ERK1/2 module via PKC–C-Raf signaling axis in Cos-7 of MKK4 as well as MKK7 (Yamauchi et al., 1999). and CHO cells (Hawes et al., 1995). In contrast, Although bg-activation of MKK4 proceeds through Gq-coupled LPA- or bradykinin receptors activate Rho and CDC42, its activation of MKK7 involves Rac ERK1/2 via calcium–calmodulin-dependent pathway (Yamauchi et al., 1999). Although the physiological involving Pyk2, Srcand Ras (Dikic et al., 1996). In significance is not known, it has been shown that HEK293 cells, a1-adrenergic receptor-mediated activa- bg-preferentially activates MKK4 in HEK293 cells. tion of ERK1/2 appears to involve PKC- as well as The signaling network that is being unraveled by these Ca2 þ -mediated pathways (Della Rocca et al., 1997). studies indicates that both Gai and Gbgi activate JNK In addition to these typical Gaq-mediated pathways, it module via distinct subunit-specific small GTPases has also been shown that Gaq can activate ERK through 2 þ (Figure 2). However, in both Gai-andGbgi-mediated a novel mechanism involving a DAG- as well as Ca - activation of JNK, a tyrosine kinase is involved in the dependent Rap-1-GEF (Guo et al., 2001). Using a activation of the respective small GTPases (Yamauchi variant of PC12 cells, PC12D, it has been shown that

Oncogene G Proteins and MAPKs ZG Goldsmith and DN Dhanasekaran 3128

PI3K α q βγβγ

PLCβ

PLCβ DAG PKC Ca++ Ca++-CAM

TK ? CaDAG- Pyk2 GEFI C-Raf

MEK-1/2 Src ERK-1/2 MEKK1 Shc p130CAS Rap1 MKK4/7 CRKII

SO JNK S MEKK2/3 DOCK B-Raf MEK5 180 C3G ERK5 MEK-1/2 Ras ERK-1/2 R Rac -Ras C-Raf

MEK-1/2 MAP3K ERK-1/2 MAP3K MKK3/6 MKK 4/7 p38MAPK JNK

Figure 3 Gq regulation of MAPK network. Gaq stimulates ERK, JNK, p38MAPK and ERK5 modules via PKC- as well as Ca2 þ -dependent pathways as illustrated (see text for details). In addition, the bg-subunits can initiate similar pathways through their ability to activate PLCb and/or PI3K. The dashed lines indicate the pathways that need to be further characterized.

m1-muscarinic receptor stimulation of ERK1/2 modules Gq stimulation of JNK is independent of Ras but dependent on Gaq-stimulated In contrast to the major role of the a-subunits of the Gq PLCb. More interestingly, it has been shown that family in activating the ERK module, the a- as well as 2 þ Gq-mediated release of DAG and Ca leads to the the bg-subunits of Gq plays determinant roles in the activation of a calcium- and diacylglycerol-regulated activation of JNK module (Coso et al., 1995, 1996; guanine nucleotide exchange factor, CalDAG-GEFI Mitchell et al., 1995; Zohn et al., 1995; Nagao et al., that can stimulate Rap-1, which in turn, can activate 1998). Receptors that couple to Gq such as the m1- B-Raf and the downstream ERK1/2 module (Guo et al., muscarinic (Coso et al., 1995), m3-muscarinic (Wylie 2001). Thus, Gaq-mediated signaling to ERK1/2 module et al., 1999), angiotensin II (Zohn et al., 1995), a1 appears to involve three distinct mechanisms: (1) adrenergic(Ramirez et al., 1997) thrombin (Shapiro PKC-mediated activation of C-Raf; (2) Ca2 þ -Pyk2- et al., 1996) and endothelin-1 (Shapiro et al., 1996) Src-dependent activation of Ras; and (3) Ca2 þ – receptors have been shown to potently stimulate the DAG-stimulated Rap1-mediated activation of B-Raf. JNK module. The findings that Gaq activates MEKK1 As Gaq/11 activation results in the generation of both and MEKK1 deficiency disrupts Gaq-mediated activa- DAG and IP3, all of these mechanisms need not be tion of JNK suggest that MEKK1 is a primary signaling mutually exclusive. It should be noted here that since bg- hub for the activation of JNK by Gaq (Minamino et al., subunits disassociated from Gq/11-coupled receptors can 2002). However, the molecular mechanism(s) through also stimulate PLCb, it is possible that they also which Gq communicates to the JNK module appears to contribute to Gq/11 signaling to ERK1/2 modules in a be cell-type specific. Using ectopic expression of Gq- similar manner. However, it has been noted that coupled m1-muscarinic receptors in NIH3T3 (Coso Gq/11-coupled receptors primarily use the DAG/ et al., 1995), Rat1a (Mitchell et al., 1995), COS-7 (Coso PKC- and/or IP3/Ca2 þ signaling pathways, rather than et al., 1996) or HEK293 cell (Nagao et al., 1998), it has bg-subunits, to activate ERK1/2 modules (Blaukat been shown that the stimulation of these receptors lead et al., 2000). A footnote here is that in some cellular to the potent activation of JNK module. While, Gq- contexts, as in the case of the activation of ERK1/2 coupled m1-muscarinic receptor activation of JNK does by Gq-coupled oxytocin receptors, the bg-disassociated not involve the PLC-PKC axis in NIH3T3 cells (Coso from Gq activates ERK1/2 through the transactivation et al., 1995), studies with COS-7 cells indicated that the of receptor tyrosine kinase (Zhong et al., 2003). activation of JNK by m1-muscarinic receptor involves a

Oncogene G Proteins and MAPKs ZG Goldsmith and DN Dhanasekaran 3129 pathway initiated by the Gbgq-involving Ras and Rac-1 Gq stimulation of p38MAPK (Coso et al., 1996). In contrast, studies using Rat1a cells Similar to its role in the activation of the JNK module, have shown that m1-muscarinic receptor activation of different Gq-coupled receptors have been shown to 2 þ JNK by Gaq involves a Ca -dependent mechanism activate the p38MAPK module (Kramer et al., 1995; (Mitchell et al., 1995), thereby suggesting a role for the Krump et al., 1997; Yamauchi et al., 1997). Further Gaq-PLC-IP3 signaling axis. Likewise, it has been studies have established that Gq can activate p38MAPK shown that Gaq-coupled angiotensin II receptor activa- module through a- as well as the bg-subunits (Yamauchi tion of JNK proceeds through such Ca2 þ -dependent et al., 1997, 2001; Nagao et al., 1998; Huang et al., signaling, possibly involving a PLC-IP3-based mechan- 2004). The basic mechanism through which Gaq ism (Zohn et al., 1995). Interestingly, such Ca2 þ - activates p38MAPK is not different from the one dependent signaling has also been shown to be sensitive involved in the activation of the JNK module. The 2 þ to genistein, suggesting a role for Ca -dependent PLC–DAG–PKC signaling arm of Gaq along with the tyrosine kinase phosphorylation (ibid). In addition, a activation of Src or an Src-like tyrosine kinase has been direct role for PKC in the activation of JNK has also shown to be involved in Gaq-mediated activation of been established. In HEK293 cells, it has been shown p38MAPK (Nagao et al., 1998; Yamauchi et al., 2001). that the expression of the constitutively activated Thus, the signaling pathways mediated by Gaq involve mutant of Ga11 results in the activation of JNK through PLC-PKC-dependent activation of Src followed by Src a pathway involving PKC as well as Srcfamily of activation of the Rho family of GTPases leading to the tyrosine kinases (Nagao et al., 1998). activation of p38MAPK module. Consistent with this The general mechanism emerging from these studies is view, it has been shown that Gaq activates MKK3 or that Gq can use both the a- as well as bg-subunits MKK6, the MAP2Ks of p38MAPK module, via involving the PLC–DAG–PKC as well as the PLC-IP3- CDC42, Racand Rho (Yamauchi et al., 2001). The Ca2 þ signaling arms to activate JNK. Src- or an Src-like observations that the Rac-specific GEFs Tiam-1 and kinase appears to be involved in the activation of JNK Ras-GRF, both of which can be activated by Src, are in in both the pathways (Zohn et al., 1995; Nagao et al., physical complex with MKK3 and p38MAPK (Buchs- 1998). Therefore, PKC activation of Src and subsequent baum et al., 2002) provides partial evidence for such a Src-mediated activation of Rac through a specific GEF pathway that can be activated by Gaq. appears to be involved in Gaq-mediated activation of In contrast to the role of PLC-PKC in Gaq-mediated JNK via the pathway involving PKC, Srcand Rac pathways, the bg-pathways derived from the Gq hetero- (Servitja et al., 2003). Although such a linear pathway trimer involves a PLC-independent mechanism(s) invol- has not been established in a specific single cellular ving Rho family of GTPases and a tyrosine kinase that context, the previous findings that PKC can readily remains to be identified (Yamauchi et al., 2001). In activate Src through direct phosphorylation (Gould addition, it has been shown that Gq-derived bg-subunit et al., 1985; Moyers et al., 1993), Src can activate Rac- can activate p38MAPK via a pathways involving Rap-1 specific GEFs (Servitja et al., 2003) and such activation (Huang et al., 2004). It is quite likely that the activation of Raccanstimulate the JNK module (Crespo et al., of Rap-1 by bg-subunits involves a Pyk2-Src-C3G 1996; Michiels et al., 1997) strongly points to such a pathway similar to the one involved in the activation pathway. of JNK (Mochizuki et al., 2000). Although the mechan- Activation of JNK module by the PLC-IP3-Ca2 þ arm ism by which Rap-1 activates p38MAPK module 2 þ of Gaq signaling has been shown to involve a Ca - is not fully elucidated, the findings that bg-pathway dependent activation of Pyk2 (Tokiwa et al., 1996; can stimulate C3G (Mochizuki et al., 2000), and C3G Blaukat et al., 1999), Pyk2-induced activation of Src, can stimulate Rap-1 (Gotoh et al., 1995) as well as Src-mediated phosphorylation of p130CAS and the MLK3/DLK family of kinases (Tanaka and Hanafusa, subsequent association of p130CAS and the adaptor 1998) and MLK3 can activate p38MAPK (Chadee and protein CrkII (Blaukat et al., 1999; Kodama et al., Kyriakis, 2004; Kim et al., 2004) points to MLK family 2003). Interestingly, the p130CAS–CRKII complex has of MAP3Ks as a possible link between Rap-1 and been shown to stimulate the JNK module via Rac-1 p38MAPK modules. (Dolfi et al., 1998; Girardin and Yaniv, 2001) presum- ably through the activation of Rac-1 by the Rac-GEF, DOCK180 (Kiyokawa et al., 1998) or R-Ras by the Gaq stimulation of p38MAPKg/ERK6 module R-Ras/Rap-1-GEF, C3G (Mochizuki et al., 2000). p38MAPKg, an isoform of p38MAPK that shows 2 þ Therefore, the signaling from Gaq via PLC-IP3-Ca distinct expression and activation profiles, is character- is likely to involve Rac-1- or R-Ras-dependent mechanisms, ized as ERK6 (Lechner et al., 1996; Kumar et al., 1997; both of which can lead to the activation of JNK module Wang et al., 2000). Similar to the p38MAPK module, by stimulating JNK-specific MAP4Ks such as MLK3 p38MAPKg/ERK6 is activated by the MAP2Ks, (Teramoto et al., 1996; Tanaka and Hanafusa, 1998). MKK3 and MKK6 (Kumar et al., 1997; Wang et al., Since bg-subunits can stimulate PLC-b and subse- 2000). It has also been shown that the small GTPase quent IP3-mediated Ca2 þ release/influx, it should be Rho activates p38MAPKg/ERK6 via its stimulatory noted here that the bg-signaling, leading to the activa- interaction with PKN, which can activate the MLK-like tion of JNK via Ras and Rho family of GTPases, kinases that act as MAP3Ks of this module (Wang et al., involves a pathway similar to the one described for Gaq. 2000; Gross et al., 2002; Takahashi et al., 2003). Initial

Oncogene G Proteins and MAPKs ZG Goldsmith and DN Dhanasekaran 3130 evidence that Gq-mediated pathways are involved in the neoplastictransformation of fibroblast celllines (Chan activation of p38MAPKg/ERK6 came from the obser- et al., 1993; Jiang et al., 1993; Xu et al., 1993; Vara vation that m1-receptor-expressing NIH3T3 cells Prasad et al., 1994; Voyno-Yasenetskaya et al., 1994; demonstrated the activation of ERK6/p38MAPKg Dhanasekaran and Dermott, 1996; Liu et al., 1997). In in response to carbachol (Marinissen et al., 1999). In view of the findings that constitutively active Ga12 was analysing the role of PAR-1 receptors in stimulating isolated as the transforming ‘oncogene’ from the Ewings c-Jun expression and oncogenic transformation, the sarcoma cell line (Chan et al., 1993) and that Ga12 ability of Gaq to activate p38MAPKg/ERK6 was potently stimulates the neoplastictransformation of characterized. It appears that the activation of ERK6 fibroblast cell lines (Chan et al., 1993; Jiang et al., 1993; is mediated primarily by the a-subunit rather than the Xu et al., 1993; Vara Prasad et al., 1994; Voyno- bg-subunit (Marinissen et al., 2003). Since Gaq can Yasenetskaya et al., 1994), it has been designated as stimulate Rho-GEF proteins such as leukemia- ‘gep’ oncogene (Xu et al., 1994; Gutkind et al., 1998). In associated Rho-GEF (LARG) (Booden et al., 2002), it addition, the expression of these a-subunits has been is very likely that Gaq-mediated activation of the shown to stimulate mitogenicresponses in many p38MAPg/ERK6 module involves Rho-dependent different cell lines (Aragay et al., 1995; Denis-Henriot activation of PKN and subsequent stimulation of et al., 1998; Coffield et al., 2004). Further analyses have MLK-family of MAP3Ks followed by MKK3/6 and revealed that these a-subunits can regulate cell prolife- p38MAPKg/ERK6 (Wang et al., 2000; Marinissen et al., ration (Aragay et al., 1995; Denis-Henriot et al., 1998; 2001, 2003; Gross et al., 2002; Takahashi et al., 2003). In Radhika and Dhanasekaran, 2001; Coffield et al., 2004; this context, it should be noted that since p38MAPKg/ Kumar et al., 2006), differentiation (Jho and Malbon, ERK6 is activated by MKK3 and MKK6, which are 1997), apoptosis (Althoefer et al., 1997; Berestetskaya also involved in the activation of other p38MAPK- et al., 1998), as well as cell migration (Offermanns et al., isoforms, it could also be regulated by the mechanisms 1997; Radhika et al., 2004; Dhanasekaran, 2006; Shan outlined in the previous section. et al., 2006) in a context-specific manner. A search for the underlying signaling mechanism(s) identified that G stimulation of ERK5 G12 proteins strongly stimulate JNK activity in a variety q of cells (Prasad et al., 1995; Collins et al., 1996; Voyno- G -mediated signaling has also been shown to regulate q Yasenetskaya et al., 1996; Jho et al., 1997; Nagao et al., the ERK-5 module via MEK5 (Marinissen et al., 1999). 1999; Arai et al., 2003; Marinissen et al., 2003), and the Studies using the transient expression of G -coupled q inhibition of which could abrogate Ga -mediated G1 muscarinic receptors have identified that the G - 12/13 q to S phase cell cycle progression (Mitsui et al., 1997). mediated signaling pathway is involved in the regulation of the MEK5–ERK-5 signaling module and subsequent transactivation of c-Jun via MEF2 family of transcrip- G signaling to JNK module tion factors (Marinissen et al., 1999). Further studies 12/13 Having 67% shared amino acid identity, Ga and Ga using a chimeric mutant of Ga and Ga , through which 12 13 q i stimulate similar responses including the activation of Ga -mediated signaling can be activated by Ga -coupled q i JNK in many cellular contexts (Radhika and Dhanase- muscarinic receptors, have established that Gaq and not Gbg is involved in the activation of ERK5 (Fukuhara karan, 2001). However, the mechanism through which q these a-subunits couple to the JNK module appears to et al., 2000b). Although the mechanism by which Ga is q be cell-type or physiological context dependent. In these coupled to the MEK5–ERK5 module is not fully different contexts, Ga and Ga activate the JNK understood, initial studies have identified that it is not 12 13 module via the small GTPases Ras, CDC42, Racor Rho through Ras, CDC42, Racor Rho (Fukuhara et al., (Figure 4). Although the activated mutant of Ga 2000b). However, the observation that the MAP3Ks 12 stimulates JNK via Ras in NIH3T3 cells, it involves MEKK2 and MEKK3 can interact and activate MEK5 Rac1 in 1321N astrocytoma and HEK293 cells (Collins (Sun et al., 2001) suggests the possible role of these et al., 1996). In Cos-7 cells, it has been shown to involve MAP3Ks in this pathway. In conjunction with the CDC42 (Voyno-Yasenetskaya et al., 1996). All of these findings that Rap-1 is involved in the activation of ERK5 via MEKK2 (Wang et al., 2006a) and G can small GTPases appear to transmit signals to the JNK q module via MEKK1, as the expression of dominant activate Rap-1 via the Rap-1-GEF CG3 or CalDAG- negative MEKK1 has been shown to inhibit Ga - GEFI as discussed in the previous sections, it is likely 12 mediated activation of JNK in all of these instances that Ga , via Rap-1, activates the MEKK2–MEK5– q (Collins et al., 1996; Voyno-Yasenetskaya et al., 1996). ERK5 module. However, these earlier events involved Thus, Ga as well as Ga communicates to MEKK1, in Ga -mediated activation of ERK5 remain to be 12 13 q the upstream MAP3K of the module, via the Ras- or established. Rho family of small GTPases, presumably through specific GEFs. The identity of the specific GEFs that couple Ga12 or Ga13 to JNK module has been identified Regulation of MAPKs byG 12 only in a few instances (Lee et al., 2004). In the case of the Ras-GEF linking Ga12 to Ras, it The G12 family of G proteins, defined by the a-subunits appears to involve Shc-mediated recruitment of SOS Ga12 and Ga13, is extremely potent in inducing (Collins et al., 1997). It has been shown that Ga12

Oncogene G Proteins and MAPKs ZG Goldsmith and DN Dhanasekaran 3131

Ras α 12/13 Rac

CDC42 α α 12 13 α Rho 12

Raf MAP3K MEKK2/3 MAP3K MAP3K Raf MEK-1/2 MKK4/6 MEK5 MKK3/6 MKK3/6 MEK-1/2 ERK-1/2 JNK ERK5 p38MAPK p38MAPK ERK-1/2

Figure 4 G12/13 regulation of MAPK networks. Ga12 as well as Ga13 can potently activate JNK module by stimulating Ras as well as Rho family of GTPases. In some cells, Ga12/13 can weakly stimulate ERK1/2 in a Ras-dependent manner, whereas in others they attenuate ERK1/2 activation at the levels of MEKs. In addition, both Ga12 and Ga13 can activate ERK5 through hitherto uncharacterized mechanism. In addition, Ga12 and Ga13 can regulate different MAPKs through pathways unique to them (dashed lines). While Ga12 inhibits p38MAPKs in some cell types at the levels of MKKs, Ga13 stimulates p38MAPKs including p38MAPKg/ ERK6 via MKK3/6 in some cells.

stimulates Shc-phosphorylation in 1321N1 cells, in Rho-GEF involved in linking Ga12 to Rho in an Src- which it stimulates JNK via Ras-dependent mechanism dependent manner and subsequent activation of JNK (Collins et al., 1996, 1997). Therefore, it has been module. However, it is also possible that Ga12 stimulates suggested that Shc-Grb2-recruited SOS is involved in Rho-mediated activation of JNK module via other Src- stimulating Ras and subsequently JNK in these cells responsive Rho-GEFs. In the case of Ga13, it has been (Collins et al., 1997). Although the kinase involved in shown that it can physically interact with p115RhoGEF stimulating the phosphorylation of Shchas not been and stimulate its GEF activity toward RhoA (Hart identified, considering the findings that Ga12 can et al., 1998). Consistent with this observation, Rho- stimulate several tyrosine kinases such as Src (Nagao dependent activation of JNK has been shown to involve et al., 1999), Fak (Needham and Rozengurt, 1998; p115RhoGEF in P19 cells (Lee et al., 2004). Notwith- Chikumi et al., 2002) and Tecfamily of kinases (Mao standing the small GTPase utilized, the MAP3K et al., 1998; Jiang et al., 1998), it possible that one of involved in Ga12/13-mediated signaling appears to these kinases may be involved in stimulating the involve MEKK1 (Collins et al., 1996; Voyno-Yasenets- phosphorylation of Shc. In this context, the observa- kaya et al., 1996; Wang et al., 2002). In some contexts, tions that Ga12 can stimulate BTK, a member of Tec the JNK module activated by Ga12/13 involves ASK1 family of kinases (Jiang et al., 1998) and BTK can (Berestetskaya et al., 1998), MEKK-2 or MEKK4 as activate JNK via Shc-Grb2-mediated activation of Ras well (Wang et al., 2002; Lee et al., 2004). Generally, the (Deng et al., 1998), make BTK or one of its family MAP3Ks activate MKK4 as well as MKK7, the JNK- members an attractive candidate in this pathway. The specific MAP2Ks. However, it has been observed that identities of the GEFs that link Ga12/13 to downstream Ga12-mediated activation of JNK preferentially involves Racand CD42 are largely unknown. MKK7 (Dermott et al., 2004), whereas Ga13- as well as In addition to Ras, Racand CDC42, it has also been bg-mediated activation appears to involve MKK4 shown that Ga12 stimulates JNK via RhoA (Nagao (Yamauchi et al., 1999; Wang et al., 2002; Lee et al., et al., 1999; Arai et al., 2003). In HEK293 cells, it has 2004). Although, the functional significance of these also been shown that Src-dependent stimulation of differential preferences for specific MKKs is largely not RhoA is involved in Ga12-mediated activation of JNK known, it should be noted here that the dual specificity (Nagao et al., 1999). The findings that inhibition of Src kinases MKK4 and MKK7 activate JNK by phosphory- by treating the cells with PP2 or expressing the lating two different amino acid residues. MKK4 has dominant negative mutant of Srcabrogated the ability been shown to preferentially phosphorylate Tyr185, of Ga12 to stimulate such activation suggested that an whereas MKK7 phosphorylates Thr187 of JNK1 Src-mediated GEF activity is involved in linking Ga12 to (Fleming et al., 2000). It has been proposed that both Rho-mediated activation of JNK module (Nagao et al., MKK4 and MKK7 are required for the maximal 1999). Although the mechanism by which Ga12 signals activation of JNK1 (Yang et al., 1997; Lawler et al., to Srcis not fully understood, the ability of G a12 to 1998; Lisnock et al., 2000). Since Ga12 appears to stimulate the GEF activity of tyrosine-phosphorylated preferentially stimulate MKK7 (Dermott et al., 2004), LARG is quite significant (Fukuhara et al., 2000a; whereas Ga13 stimulates MKK4 (Wang et al., 2002; Lee Suzuki et al., 2003). LARG may thus be a candidate et al., 2004), it is likely that the Ga12- and Ga13-mediated

Oncogene G Proteins and MAPKs ZG Goldsmith and DN Dhanasekaran 3132 response requires only a partial activation of JNK from directly stimulate phosphatase PP5 (Yamaguchi et al., the respective MAP2K signaling nodes. It is likely that 2002) as well as PP2A (Zhu et al., 2004), it is likely that such partial activation of JNK along with other either or both of these phosphatases play a role in signaling inputs, such as the activation of ERK module attenuating p38MAPK module. However, such a role and attenuation of p38MAPK module, are prerequisites for these proteins in Ga12-mediated attenuation of for Ga12-mediated cell proliferation of NIH3T3 cells or p38MAPK module remains to be clarified. Likewise, Ga13-mediated P19 cell differentiation in which the whether the downregulation of p38MAPK by Ga12 is differential recruitment of MAP2Ks have been observed seen in other cell types remain to be established. (Dermott et al., 2004; Wang et al., 2002; Lee et al., However, such differential regulation of JNK and 2004). p38MAPK modules is likely to have a significant role in the potent mitogenicactivity of G a12 seen in diverse cell types (Chan et al., 1993; Jiang et al., 1993; Xu et al., Ga attenuation of ERK1/2 12/13 1993; Vara Prasad et al., 1994; Voyno-Yasenetskaya In contrast to their potent activation of the JNK et al., 1994; Aragay et al., 1995; Dhanasekaran and module, Ga and Ga attenuate the activation of 12 13 Dermott, 1996; Liu et al., 1997; Denis-Henriot et al., ERK1/2 module in a cell-type-dependent manner 1998; Coffield et al., 2004). It is likely that the weak (Voyno-Yasenetskaya et al., 1996). Ras-mediated albeit stimulation of ERK activity and the decisive down- weaker stimulation of ERK1/2 has been shown to be regulation of p38MAPK in addition to the potent critically required – along with Ras-/Rac-mediated stimulation of JNK module provide the multiple potent stimulation of JNK activity – for Ga -induced 12 signaling inputs required for Ga -mediated cell pro- G1–S phase cell cycle progression of NIH3T3 cells 12 liferation. (Mitsui et al., 1997). However, the ectopic expression of Ga12/13 stimulated JNK activity with a concomitant inhibition of ERK1/2 pathway (Voyno-Yasenetskaya Ga13 stimulation of p38MAPg/ERK6 et al., 1996). Further analysis has identified MEK as the In contrast to Ga12, but similar to Gaq,Ga13 has been locus at which Ga12/13 exerts its inhibitory effects on shown to stimulate the p38MAPKg/ERK6 module ERK module. Although the underlying mechanism as (Marinissen et al., 2003). Considering the inhibitory well as the physiological significance of such regulation effect of Ga12 on MKK3/MKK6, it is more likely that is not fully known, recent studies have demonstrated Ga12 exerts similar attenuation of p38MAPKg/ERK6, that Ga12/13 can interact with PP5 to stimulate its at least in fibroblasts such as NIH3T3 cells. However, activity (Yamaguchi et al., 2002) and PP5 is involved in the transient expression of Ga13 has been shown to the downregulation of ERK module by dephosphory- stimulate p38MAPKg/ERK6 activity in NIH3T3 as well lating Raf-1 at Ser-338 (von Kriegsheim et al., 2006). as HEK293 cells. It has been suggested that ERK6 Therefore, although it remains to be established, it is stimulation of ATF2 and MEF2 is required for the possible that Ga12/13-mediated activation of PP5 is complete transactivation of c-Jun and the resulting involved in the inhibition of ERK1/2 modules. cellular transformation mediated by Ga13-coupled PAR-1 receptors (Marinissen et al., 2003). It has been identified that Rho is involved in the upstream stimula- Inhibition of p38MAPK by Ga 12 tion of the ERK6 module via PKN family of protein The expression of Ga has also been shown to attenuate 12 kinases (Marinissen et al., 2001, 2003). As Ga can the activation of the p38MAPK module (Dermott et al., 13 stimulate Rho via different GEFs, the mechanism by 2004). The inhibition was found to be at the level of the which Ga transmits signals to p38MAPKg/ERK6 is three MAP2Ks involved in the activation of p38MAPK, 13 likely to involve Rho and Rho-activated PKN. Since the namely MKK4, MKK3 and MKK6. It is interesting MAP3Ks such as MLK-like MAP triple kinase (Taka- that Ga stimulates JNK-specific MKK7 while inhibit- 12 hashi et al., 2003) or MLK-related kinase (Gross et al., ing MKK4, which is common to both JNK and 2002) can regulate ERK6 via MKK3/6 (Wang et al., p38MAPK. Thus, Ga appears to block decisively all 12 2000; Marinissen et al., 2001, 2003; Gross et al., 2002; the possible signaling routes that lead to the activation Takahashi et al., 2003), it is likely that Ga -activation of p38MAPK. However, it should be noted here that the 13 involves a phospho-relay module involving these inhibition of p38MAPK appears to be specific for Ga 12 upstream kinases. as the expression of Ga13 in NIH3T3 cells stimulates both JNK as well as p38MAPK activities (Marinissen et al., 2003). Although the molecular basis for such Ga12/13 stimulation of ERK5 selective inhibition of an MAPK at the levels of multiple In analysing the mechanism by which PAR-1 receptors, MAP2Ks by distinct a-subunits of the G12 family is not which can couple to both Gq and G12-family of G clear at present, at least two possible mechanisms can be proteins, activate ERK5, it was identified that the speculated: one involving the inhibition of p38MAPK- a-subunits of G12 as well as G13 are involved in the specific MKKs, presumably through the activation of a regulation of ERK5. Since activation of ERK5 by specific phosphatase, and the other involving a specific constitutively active Ga12 and Ga13 was insensitive to scaffolding protein that distinctly couples Ga12 to the the coexpression of the dominant negative mutants of MKK7–JNK module, while attenuating the MKK3/6/4- Ras, CDC42, Racor Rho, the activation appears to p38MAPK module. Since Ga12 has been shown to involve an alternate GTPase or mechanism (Fukuhara

Oncogene G Proteins and MAPKs ZG Goldsmith and DN Dhanasekaran 3133 et al., 2000b). An attractive candidate is Rap-1, which a G-protein-independent as well as G-protein-dependent can stimulate MEKK2 and hence the MEKK2–MEK5– pathways (Conway et al., 1999). In the G-protein ERK5 module (Wang et al., 2006a). However this independent, classical pathway, signaling to ERK possibility as well as the mechanism by which Ga12/13 involves PDGF-activated PDGFR autophosphoryla- communicates to Rap-1 needs to be experimentally tion on tyrosine residues, recruitment of adaptor verified. proteins and activation of p42/p44 MAPK. However, the G-protein-dependent pathway appears to involve Gi and b-arrestin-mediated endocytosis. In this pathway, endocytitic internalization of Gi-activated GPCRs along Receptor tyrosine kinases in G-protein-mediated with Gi-activated c-Src, PDGFR, ERK module and activation of MAPKs Grb-2 is facilitated by arrestin and dynamin II. It has been speculated that the close proximity and the possible In addition to the signaling pathways leading to the activation of Raf-1 by Src promotes the activation of regulation of MAPK modules by different G proteins, ERK module in endosomes (Waters et al., 2005). GPCRs have been shown to regulate ERK1/2 modules Further studies should define the role of such network- through the transactivation of RTKs. Such a novel role ing between RTKs and G proteins in specific cellular for EGFR in the activation of ERK by GPCRs was responses. identified with the observation that the stimulation of Rat1a cells with GPCR agonists including endothelin, LPA and thrombin stimulated the phosphorylation of EGFR and Her2 along with the activation of ERK2 Scaffold proteins in G-protein signaling to MAPKs (Daub et al., 1996). The findings that the treatment of As discussed in previous sections, diverse GPCRs, these cells with the EGFR inhibitor tyrphostin AG1478 or the expression of a dominant negative EGFR mutant through the a- as well as the bg-subunits of the four classes of G proteins, modulate the activities of different inhibited such ERK activation indicated the critical role MAPK modules. Interestingly, as can be seen in the case of EGFR in the activation of ERK by GPCRs (Daub et al., 1996). Further analyses revealed that EGFR plays of PAR- or thrombin receptors, a specific GPCR may recruit a-orbg-subunits from different classes of G a determinant role in GPCR-mediated activation of the proteins to activate an MAPK module in a cell type- or ERK module in a variety of cell types (Daub et al., 1997; context-specific manner. Even the individual a-orbg- Zwick et al., 1997; Eguchi et al., 1998). Primarily, G - i subunit may activate a specific MAPK module through and G -coupled receptors transactivate EGFR through q more than one mechanism in a context-specific manner. a novel mechanism involving ‘ecto-domain shedding’ In addition, the signaling molecules providing these and subsequent generation of soluble EGF-like growth mechanisms such as the small GTPase, their cognate factors that can stimulate EGFRs (Prenzel et al., 1999). GEFs, and the downstream kinases, are often shared by Recently, the molecular basis for GPCR-mediated different Ga-orbg-subunits activating distinct MAPK transactivation of EGFR has been summarized (Ohtsu et al., 2006). The transactivation of EGFR by GPCRs modules. So the major question is how do these distinct pathways from different G proteins elicit distinct involves both intracellular as well as extracellular functional responses involving specific MAPKs with mechanisms. Thus, EGFR transactivation by G -or i little or no signaling crosstalk with each other. Although G -coupled receptors has been shown to require the q studies from yeast have pointed out that specific scaffold intracellular generation of Ca2 þ (Zwick et al., 1997; proteins such as Ste5 can facilitate such context-specific Eguchi et al., 1998; Murasawa et al., 1998; Soltoff, 1998; linking of G-protein signaling to downstream MAPK Iwasaki et al., 1999), and activation of PKC, Src and modules (Herskowitz, 1995; Elion, 2001; Elion et al., Pyk2 (Gschwind et al., 2001). These pathways appear to 2005), the evidence for such scaffold proteins in converge on the activation of a family of a disintegrin mammalian G-protein signaling to MAPKs emerged and metalloprotease (ADAM) and/or matrix metallo- protease (MMP) proteins through phosphorylation as only recently. Of these scaffold proteins, some of them such as the b-arrestins play a catalytic as well as well as protein–protein interactions (Higashiyama and anchoring role in linking different GPCRs to MAPK Nanba, 2005; Ohtsu et al., 2006). The activated single transmembrane ADAMs, specifically ADAM-10, -12, signaling, whereas others such as MAP kinase organizer (MORG) and JLP play an anchoring role by tethering -15, -17, cleave the ectodomains of membrane-bound specific G proteins and MAPK modules in a context- EGFR ligands such as HB-EGF to generate soluble specific manner (Garrington and Johnson, 1999; Morrison EGF ligands that can activate EGFRs (Gschwind et al., and Davis, 2003; Kolch, 2005). 2001; Schafer et al., 2004; Higashiyama and Nanba, 2005; Ohtsu et al., 2006). Although these studies have shown the role of EGFR Catalytic role of b-arrestin in GPCR signaling to ERKs in G-protein signaling, it has also been shown that The arrestin family of proteins was initially identified signaling by RTKs to the ERK module can be enhanced to be involved in the desensitization of GPCRs by G proteins through a mechanism involving by facilitating their internalization (Freedman and b-arrestin. For example, it has been shown that the Lefkowitz, 1996; Kohout and Lefkowitz, 2003). Signaling activation of the ERK module by RTK ligands involves by agonist-activated GPCRs is terminated by the

Oncogene G Proteins and MAPKs ZG Goldsmith and DN Dhanasekaran 3134 GRK-family of serine/threonine kinases that phosphory- MORG-1 suppressed ERK-1 activation by FBS (which late the receptors at their C-termini (Freedman and contains several ligands including LPA that can activate Lefkowitz, 1996). b-arrestins bind to such phospho- the respective GPCRs), it did not have any effect on rylated receptors and facilitate their internalization EGF-stimulated ERK response. Thus, MORG-1 that by endocytosis via clathrin-coated pits (Shenoy and can associate with the MAP3K, MAP2K, as well as the Lefkowitz, 2003). Interestingly, it has been shown that MAPK components of ERK1/2 module, appears to b-arrestin-1 can bind to both GRK-phosphorylated provide a scaffolding function specifically for GPCRs receptors, such b2-AR, as well as c-Src, targeting them such as LPA receptors. At present, it is not known to clathrin-coated pits where the complex acts as whether such a role of MORG1 involves its physical signaling hub for the activation of ERK1 and ERK2 interaction with specific GPCRs, Ga-orGbg-subunits. (Luttrell et al., 1999). In addition, it has been shown that Further studies should define the molecular mechanism b-arrestin-2 can sequester components of the ERK by which MORG-1 links GPCR signaling to ERK1/2. module such as Raf-1, MEK1/2 and ERK1/2 via its interaction with Raf-1 (Luttrell et al., 2001). The b- Role of JLPin linking G a12/13 to JNK module arrestin scaffold containing the components of the ERK JLP was initially identified as a Leucine Zipper domain module and the receptor are internalized via clathrin- containing protein that can interact with Max (Lee coated pits into endosomal vesicles where the receptor et al., 2002). In addition, it has been observed that can activate the ERK module (Lefkowitz and Shenoy, JLP can interact with JNK, p38MAPK, MKK4 2005). Although the mechanism by which the inter- and MEKK3. Furthermore, it was observed that the nalized GPCR can activate the ERK module is yet to expression of JLP potentiated cytotoxic stress-mediated be elucidated fully, it is possible that the other activation of JNK (Lee et al., 2002). Recent studies have co-internalized intermediary molecules such as Src play shown that JLP can interact with Ga as well as Ga a role in providing such a link. Interestingly, it has been 12 13 and provide a scaffolding role for JNK activation by noted that the ERK module activated by b-arrestin has these a-subunits (Kashef et al., 2005). It has been slower kinetics of activation than that mediated by G demonstrated that JLP interacts with the a-subunits of proteins (Lefkowitz and Shenoy, 2005). In addition, it the G family through the C-terminal region of JLP, has been observed that the ERK module activated by 12 spanning amino acids 1165–1307. Using the expression b-arrestin is not involved in nuclear signaling (DeFea of this Ga interacting domain or siRNA-mediated et al., 2000; Tohgo et al., 2002). Thus, b-arrerestin-2- 12/13 silencing of JLP, it was demonstrated that the disruption mediated activation of ERK module by different of the JLP–Ga interaction leads to the attenuation of GPCRs appears to be involved in the cytosolic responses 13 LPA- as well as Ga -mediated activation of JNK. that require slower but persistent activation of ERKs 13 Furthermore, it has been observed that silencing JLP (DeFea et al., 2000; Tohgo et al., 2002; Lefkowitz and inhibits retinoicacid-mediated endodermal differentia- Shenoy, 2005). tion of P19 cells (Kashef et al., 2006), which is critically In addition to the activation of the ERK module, dependent on Ga -mediated activation of JNK (Jho b-arrestin-2 also appears to be involved in regulating 13 et al., 1997; Wang et al., 2002; Lee et al., 2004). In this the JNK module. Using yeast two hybrid screening, it context, it is significant to note that JLP is the first was identified that b-arrestin-2 can interact with JNK3 known mammalian scaffold protein, albeit similar to (McDonald et al., 2000). Further analyses indicated that Ste5, in tethering all the signaling components from the b-arrestin-2 interacts with the MAP3K, ASK1. In proximal Ga-subunit to the downstream MAPK. Since addition, MKK-4 also can be colocalized along with the Ga12/13-interacting C-terminus of JLP is conserved b-arrestin, ASK1 and JNK3 in a complex. It has also among all the JIP family of scaffold proteins, it is been observed that angiotensin II type IA receptor possible that JIPs may also be involved in linking Ga stimulated the colocalization of b-arrestin-2 and JNK3 12/ 13 or other a-subunits to JNK or p38MAPK modules in to intracellular vesicles along with the activation of a context-specific manner. This, as well as other finer et al JNK3 (McDonald ., 2000). However, it is not clear details of the JLP scaffold, such as its interactions with whether b-arrestin plays a catalytic role in stimulating specific small GTPases and their GEFs, remains to be JNK3 in this context. established. Similarly, further studies should define the role of such scaffold proteins in Ga-orGbg-mediated activation of other MAPK modules. Role of MORG-1 in linking GPCRs to ERK1/2 pathway MORG-1 was identified as a 35 kDa protein that interacts with MEK-partner 1 proteins in a yeast two hybrid screen (Vomastek et al., 2004). Further charac- Conclusion terization of this WD-40 motif containing protein indicated that it can associate with Raf-1, B-Raf, Recent studies have contributed to a greater under- MEK1, MEK2, ERK1 and ERK2. Although the ectopic standing of the mechanisms by which the GPCR family expression of MORG1 enhanced LPA or PMA- of cell surface receptors critically regulate different mediated activation of ERK, it failed to have such aspects of cell growth via the MAPK signaling modules. stimulatory effects on PDGFR- or EGFR-mediated As the signaling nexus between different G proteins activation of ERK. In addition, although silencing and distinct MAPK modules are being realized, the

Oncogene G Proteins and MAPKs ZG Goldsmith and DN Dhanasekaran 3135 pathophysiological roles of aberrant- or asynchronous cancers (Mills and Moolenaar, 2003; Booden et al., signaling by these pathways are becoming increasingly 2004; Daaka, 2004; Boire et al., 2005). The findings that clear. However, as presented in this review, several all of these GPCRs, their cognate G proteins and the G- aspects of these pathways involved in G-protein- protein oncogenes gsp, gip2 and gep converge on the mediated MAPK activation remain to be resolved. activation of distinct MAPK modules, taken together For example, the molecular basis for context-specific with the observation that GPCR-mediated signaling signaling, the spatio-temporal organization of G-protein– pathways are well-validated drug targets, underscore MAPK signaling complexes, the effects of these path- the critical need for defining these pathways further so ways in relation to receptor tyrosine kinase- or cytokine that better therapeutictargets can be identified. receptor-mediated activation of MAPKs, and most importantly, the potential role of G-protein-regulated MAPK networks in tumorigenesis and progression Acknowledgements remain to be fully characterized. A case to the point is the identification of the potential role of LPA- and The study was supported by a grant from NIH (GM 49897). PAR-receptor-mediated signaling pathways in the gene- Critical reading of the paper by Dr Rashmi Kumar and sis and progression of ovarian, breast and prostate Mr Jake Gardner are gratefully acknowledged.

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