Transforming G proteins - Nature

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Heterotrimeric guanine nucleotide binding proteins, commonly known as G proteins form a super-family of signal transduction proteins. They are peripherally.
Oncogene (2001) 20, 1607 ± 1614 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

Transforming G proteins V Radhika1 and N Dhanasekaran*,1 1

Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania, PA 19140, USA

Heterotrimeric guanine nucleotide binding proteins, commonly known as G proteins form a super-family of signal transduction proteins. They are peripherally associated with the plasma membrane and provide signal coupling to seven transmembrane surface receptors. G proteins are composed of monomers of a, b, and g subunits. The b- and g-subunits are tightly associated. The receptors activated by the appropriate `signal', interact catalytically with speci®c G-proteins to mediate guanine nucleotide exchange at the GDP/GTP binding site of the G-protein a-subunits, thus displacing the bound GDP for GTP. The GTP bound form of the gprotein a-subunit and in some cases the free bg-subunits initiate cellular response by altering the activity of speci®c e€ector molecules. Recent studies have indicated that the asyncronous activation of these proteins can lead to the oncogenic transformation of di€erent cell types. The mechanism by which G-proteins regulate the various cell functions appear to involve a complex net-working between di€erent signaling pathways. This review summarizes the signaling mechanisms involved in the regulation of cell proliferation by these transforming G proteins. Oncogene (2001) 20, 1607 ± 1614. Keywords: G protein; oncogene; signal transduction; transformation; Ras; Rac Introduction Heterotrimeric G proteins consisting of a-, b-, and gsubunits, provide signal coupling mechanisms to seven transmembrane receptors. The G protein in its unstimulated con®guration exists as the GDP-bound a-subunit complexed with the b-and g-subunits. The band g-subunits are tightly bound and often referred to as the bg-subunit. In a typical G protein coupled signaling pathway, ligand-activated receptor catalyzes the exchange of guanine nucleotides in the a-subunit. The GTP-bound a-subunit dissociates from the receptor as well as the bg-subunit and activates its respective e€ector molecule. The free bg-subunit also activates the same or di€erent e€ector molecule(s). The hydrolysis of the bound-GTP to GDP by the intrinsic GTPase activity of the a-subunit leads to a conformational switch that results in the termination of its e€ector-interaction. The a-GDP, thus formed, reassociates with the free bg-subunit and the newly *Correspondence: N Dhanasekaran

formed a-GDP-bg heterotrimer re-enters the signaling cycle. Fine-tuning of this basic signaling unit is mediated by other signal modulators such as GPCRkinases, RGS proteins, and e€ectors with intrinsic GAP activity. Furthermore, signaling proteins such as non-receptor tyrosine kinases, protein kinase C, ADPribosylases, and lipid-transferases also play a decisive role in the pathways regulated by G proteins by covalently modifying the receptors and/or the G protein subunits. The subunits of the heterotrimeric G proteins show a wide range of heterogeneity. To date, 17 a-subunits, ®ve b-subunits, and 12 g-subunits have been identi®ed and cloned. Notwithstanding the signaling roles of bg-subunits, the heterogeneity of the a-subunits has been used to denote and classify the G proteins. Thus the a-subunits having the amino acid identity of 50% or more are grouped into four distinct classes or subfamilies namely, Gs, Gi, Gq, and G12 (Strathman et al., 1989; Strathman and Simon, 1991). Although this classi®cation is rather arbitrary, a general similarity among the members of the subfamilies can be seen. For example, the a-subunits belonging to Gs family stimulates adenylyl cyclase whereas the a-subunits from Gi-family inhibits adenylyl cyclase. The members of Gq family of proteins couple to the phospholipase c-b whereas the members of G12 family appear to be predominantly involved in the regulation of small GTPases. The role of G proteins in regulating cell growth and di€erentiation was realized with the observation that several tropic hormones activate cell proliferation and di€erentiation through cAMP-dependent pathway suggesting that Gas or Gai signaling pathway may be involved in the growth-regulation of these cells (Dumont et al., 1989). This reasoning followed by the observation that the activating mutations of the as and ai were found in a subset of endocrine tumors led to the investigation of growth altering properties of other G proteins (reviewed by Gupta et al., 1992a; Pouyssegur and Seuwen et al., 1992; Dhanasekaran et al., 1995; Dhanasekaran and Vara Prasad, 1998; Gutkind et al., 1998). With the advent of ®broblast transformation assay in which the growth promoting and transforming ability of diverse proteins can be monitored (Copeland et al., 1979) the transforming activity of several G proteins have been identi®ed (Dhanasekaran and Vara Prasad, 1998). Studies using the expression of the activated mutants of di€erent asubunits in diverse cell-types, novel mechanisms through which these a-subunits regulate cell growth and di€erentiation have been identi®ed. Of the 17 a-

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subunits that have been cloned to date, 10 of them, Gas, GaI1, GaI2, Gao, Gaq, Ga11, Ga16, Gaz, Ga12, and Ga13 have been shown to be involved in the regulation of cell growth. This review brie¯y summarizes the possible mechanisms through which these G proteins activate cell proliferation and oncogenic transformation. Although the bg subunits of G proteins do play an equally important role by interacting with the same or distinct e€ectors in the regulation of growth, since the role of bg-subunits have been excellently reviewed by Schwindinger and Robishaw elsewhere in this issue, it is not discussed here (Schwindinger and Robishaw, 2001). Gas Tropic hormones such as growth hormone releasing hormone (GHRH) and thyroid stimulating hormone (TSH) stimulate cell proliferation in pituitary and thyroid tissues through their respective Gs-coupled receptors. Conforming to this view, it was observed that the Gas-mediated signaling pathway is constitutively activated in a subset of human pituitary tumors (Vallar et al., 1987). This was followed by a search for the activating mutations in di€erent cAMP-dependent endocrine tissues. Subsequently, point mutations in the as subunit involving either arginine 201 (R201) or glutamine 227 (Q227) were identi®ed as the activating mutations involved in pituitary GH secreting tumors and thyroid hyperfunctioning adenomas (Landis et al., 1989; Lyons et al., 1990). Accordingly, the mutationally activated form of Gas is known as the gsp oncogene (derived from Gs protein). However, the gsp mutations have been detected only in pituitary tumors, thyroid adenomas and thyroid carcinomas. In these tumors, the activating mutations result in the constitutive activation of adenylyl cyclase-cAMPcAMP-dependent protein kinase (PKA) signaling pathway which leads to abnormal cell proliferation (Vallar, 1996). The role of the gsp mutations in thyroid and pituitary neoplasia was investigated using thyroidderived FRTL-5 cells (Muca and Vallar, 1994; Zieger et al., 1996; Ham et al., 1997). Expression of gsp has been observed to stimulate the adenylyl cyclase activity followed by an increased rate of DNA synthesis independent of the tropic hormone, TSH. Pituitaryderived GH3 cells expressing gsp also showed a similar increase in cAMP levels along with the activation of cell proliferation (Ham et al., 1997). It has also been shown that the persistent activation of adenylyl cyclase by expressing the A1-fragment of the cholera toxin led to an increased proliferation of FRTL-5 cells (Zieger et al., 1996). In addition, the ®nding that the injection of these cells into nude mice resulted in the formation of tumors clearly indicated the role of cAMP in TSHindependent cell proliferation and neoplastic transformation of thyroid tissue (Muca and Vallar, 1994; Zieger et al., 1996). What is the mechanism by which Gs and gsp promote cell growth? The growth promoting activity

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of Gs and its oncogenic form gsp is due to the constitutive activation of the adenylyl cyclase (AC) pathway. AC mediated increase in cAMP levels leads to the persistent activation of PKA resulting in the phosphorylation and subsequent activation of cAMP responsive element-binding protein (CREBP). CREBP binds to cyclic AMP responsive elements (CREs) to transactivate the transcription of speci®c primary response genes that initiate cell proliferation. However, it should be noted here that Gs stimulates proliferation only in the cells ± such as those present in endocrine tissues ± that are positively responsive to cAMP-PKA signaling pathway for their cell growth. By contrast, in other cells, Gas appears to have a growth inhibitory e€ect through its negative regulation of Ras-Raf signaling pathway (Burgering et al., 1993; Graves et al., 1993; Cook and McCormick, 1993; Wu et al., 1993; Sevetson et al., 1993). In these cell types, PKA activated by Gas through cAMP directly phosphorylates Raf at Ser 43 and/or Ser 671 (Wu et al., 1993; Mischak et al., 1996) thus inhibiting Raf and its downstream MEK-ERK cascade. Thus, gsp can have two opposing e€ects depending on the cells in which they are expressed (Figure 1). It should also be noted here that the oncogenic activity of gsp is very much dependent on the cell type in which it is expressed. For instance, when the oncogenic activity of gsp was assessed using Swiss 3T3 cell based transformation assay, the expression of the gsp oncogene resulted in an increase in the rate of DNA synthesis and cell proliferation that could be correlated with elevated levels of cAMP. Nevertheless, the enhanced mitogenic pathway stimulated by gsp did not lead to the transformation of these cells (Zachary et al., 1990). Gai Mutations of R179 and Q205 in Gai lead to the constitutively activated GTPase de®cient phenotype. Mutations in these residues of Gai2 have been observed in di€erent forms of tumors. While R179-mutation of Gai2 was identi®ed in ovarian sex cord stromal tumors and adrenal cortex tumors (Lyons et al., 1990), Q205mutation has been detected in non-functioning pituitary tumors (Tordjman et al., 1993). These activated mutant forms of Gai2 are denoted as gip2 oncogenes (for Gi protein-2). Expression of gip2 has been shown to induce oncogenic transformation of Rat1a cells (Pace et al., 1991; Gupta et al., 1992b). Gip2transformed Rat1a cells exhibit true transformed phenotype by exhibiting anchorage independent cell growth by forming colonies on soft agar. Furthermore, the injection of these cells has been shown to form tumors in athymic nude mice (Pace et al., 1991; Gupta et al., 1992b). Consistent with these ®ndings, inactivating gip2 function has been shown to inhibit cell growth and tumor formation (Hermouet et al., 1993; 1996). When K1735-CL19 cells expressing Gai2G204A were injected into athymic nude mice, the tumor formation was much delayed compared to the animals injected

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Figure 1 Schematic model for the regulation of cell proliferation by G12/13. The growth regulatory signals transduced by G12/13 (see text for details). GPCR, G protein coupled receptor; GEF, guanine nucleotide exchange factor; GDI, guanine nucleotide dissociation inhibitor; GAP, GTPase-activating protein; PAK, p21-activated kinase; ERK, extracellular-signal regulated kinase; MEK, mitogen-activated extracellular-signal regulated kinase kinase; MEKK, MEK kinase; JNK, Jun N-terminal kinase; JNKK, Jun N-terminal kinase kinase; NHE, Na+/H+ exchanger

with either control cells or cells expressing gip2. Furthermore, the tumors formed in these animals were reduced in size. It has also been observed that the expression of Gai2G204A in these melanoma cells inhibited their growth by 50% (Hermouet et al., 1996). Taken together, these results suggest that the gip2 oncogene, as well as its protooncogene Gai2, are involved in the onset and the propagation of many di€erent forms of tumors. The mechanism through which gip2 activates cell proliferation and oncogenesis is not fully understood. However, the ®nding that the expression of gip2 constitutively activates MEK-ERK signaling in Rat1a cells indicated that this could be the major mechanism through which gip2 regulates cell growth (Gupta et al., 1992b). Interestingly it has been observed that gip2-mediated activation of MEK-ERK pathway is independent of functional Ras (Winitz et al., 1993). Based on these observations, it has been proposed that the Ras-independent activation of MEK-ERK signaling pathway by gip2 is due to the inhibitory e€ect of gip2 on the levels of intracellular cAMP (van Biesen et al., 1996). It has been known that the expression of gip2 leads to a decrease in the cAMP levels and the resultant PKA activity in Rat1a cells (Gupta et al., 1992b,c; Lowndes et al., 1991). Since PKA is known to inhibit Raf through phosphorylation, a reduction in PKA and subsequently on MEKERK signaling pathway (van Biesen et al., 1996; Dhanasekaran and Vara Prasad, 1998). Thus, through its downregulation of AC-cAMP-PKA pathway, gip2 can upregulate MEK-ERK signaling pathway and

promote cell proliferation (Figure 1). Such a view would be consistent with the observation that the activation of ERK by gip2 is independent of Ras (Winitz et al., 1993). The activation of MEK-ERK signaling pathway couples gip2 signaling to the transactivation of TCFs in the nucleus. Based on the observation that the gip2 mutations activate cell proliferation, the growth regulating properties of the other members of Gai family was investigated. The respective a-subunits were expressed in di€erent ®broblasts to test their ability to transform the ®broblast cell lines. These studies have indicated that the GTPase-de®cient mutant of Gai1 is very similar to the gip2 oncogene in activating cell proliferation whereas the analogous activated mutant of Gai3 is not (Hermouet et al., 1993). Gaz and Gao, both of which belong to the Gi family of G proteins, have also been shown to activate mitogenic pathways in di€erent cell-types (Wong et al., 1995; Kroll et al., 1992; van Biesen et al., 1996). Since the possible mechanisms through which these a-subunits regulate cell proliferation and oncogenic transformation have been excellently reviewed separately by Ram and Iyengar (2001) and Ho and Wong (2001) in this issue, they are not discussed here. Gaq The transforming activity of Gaq has been investigated using the activating mutation of Gaq (GaqQ209L). While the expression of GaqQ209L has been shown to Oncogene

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transform NIH3T3 cells (De Vivo et al., 1992; Kalinec et al., 1992). However, GaqQ209L-induced foci formation in NIH3T3 cells was observed to be dependent upon the priming of the transfected cells with dexamethasone (De Vivo et al., 1992). Kalinec et al. (1992) also observed that the injection of GaqQ209Ltransformed NIH3T3 cells into athymic nude mice induced tumors by 1 week. Interestingly, the expression of GaqQ209L in NIH3T3 cells caused more cell death than transformed phenotype. Expression of GaqQ209L proved to be cytotoxic to 80% of the transfectants. Based on these ®ndings it has been proposed that the low levels of expression of GaqQ209L transforms the ®broblasts while higher levels of expression leads to their death. Hence the growth promoting activity of Gaq appears to be conditional upon the physiological status of the cell as well as the expression levels of the activated Gaq. Since Gaq is known to activate PI-PLCb (Wu et al., 1992; Conklin et al., 1992), the role of PLC in Gaqmediated cell proliferation has been investigated. It has been observed that the microinjection of PLC triggers transformation in NIH3T3 cells (Smith et al., 1989). In addition, it has been noticed that the expression of m1, m2, m5 muscarinic- (Gutkind et al., 1991), a1b adrenergic- or 5HT1c-receptors (Allen et al., 1991) that are known to be coupled to PLC pathways induce the oncogenic transformation of NIH3T3 cells. Taken together, these observations suggest that the activation of PLC is the underlying mechanism involved in the mitogenic and oncogenic pathways regulated by Gaq. The activation of PLC leads to cleavage of phosphatidylinositols generating inositol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG). The diacylglycerols generated from these pathways activate PKC and PKC in turn can stimulate ERK through Raf (Zou et al., 1996; Marais et al., 1998). Thus, in cell types such as CHO, it has been observed that Gaq can activate an ERKmediated proliferative pathway through a PKCdependent but Ras-independent mechanism (Figure 1). However, in other cell types such as the rat vascular smooth muscle and NIH3T3 cells, Gaq appear to activate ERK through a novel pathway involving proline-rich tyrosine kinase-2 (Pyk2) and Ras (Lev et al., 1995; Bourne, 1995). This pathway appears to be activated by the IP3 generated by the activation of PLC. IP3 increases the cellular levels of Ca2+ levels by stimulating the intra- as well as the extracellular mobilization of Ca2+. The IP3-induced increase in Ca2+ levels, presumably through a Ca2+/ calmodulin-dependent kinase, stimulate the activity of Pyk2 to activate Shc through tyrosine phosphorylation. The resultant Shc-GRB2-SOS complex stimulates Ras, thus leading to the activation of ERK (Lev et al., 1995; Bourne, 1995). Thus, Gaq can regulate growth through either a Ras-dependent or Ras-independent but PKC-dependent ERK pathway (Figure 1). Either of these pathways can couple Gaq signaling to the nuclear events through the activation of TCFs or TRE-speci®c transcription factors.

Ga12/13 Ga12 and Ga13, the members G12 family of G proteins are expressed ubiquitously in most of the tissues (Strathmann and Simon, 1991; reviewed by Dhanasekaran and Dermott, 1996). Ga12 and Ga13 have a molecular weight of 43 000 kDa and they show more than 66% amino acid identity. The observation that the deletion of Cta ± Concertina (cta) gene product of Drosophila which shows close similarity to Ga12 and Ga13 (53 ± 55%) ± disrupts Drosophila ventral furrow development (Parks and Wieschaus, 1991) suggested the possibility that Ga12 might be involved in the regulation of cell growth. A direct role for Ga12 in the regulation of cell proliferation rather came from an unrelated study focused on characterizing tumorspeci®c oncogenes. When Aaronson and his co-workers used an expression cloning method to identify the putative oncogene of soft-tissue sarcoma, Ga12 was identi®ed as the `transforming oncogene' (Chan et al., 1993). NIH3T3 cells transformed by Ga12 were characterized by their decreased doubling time, loss of saturation density of growth, and ability to form colonies on soft-agar in addition to forming tumors in athymic nude mice. Further characterization of the transforming property of Ga12 indicated that the expression of wild-type Ga12 itself was sucient to alter the growth properties of NIH3T3 cells (Chan et al., 1993). It was also observed that the `transformation' by Ga12 was dependent on the presence of serum in the growth medium since serum starvation inhibited the transforming ability of wild-type Ga12 (Chan et al., 1993). Interestingly, the expression of GTPase de®cient, activated mutant of Ga12, Ga12Q229L, can abrogate the need for the serum/agonist-dependency for transformation (Xu et al., 1993). Subsequently, the ability of the GTPase-de®cient of Ga12 (Ga12Q229L) and Ga13 (Ga13Q226L or Ga13QL) to transform various ®broblast cell lines have been documented by di€erent research groups (Xu et al., 1993, 1994; Jiang et al., 1993; Vara Prasad et al., 1994; Voyno-Yasenetskaya et al., 1994). It should be stressed here that the activated mutants of Ga12 and Ga13 are the most potent transforming a-subunits that have been tested so far. Based on these observations, Gutkind and his colleagues have designated the G12 class of a-subunits as the gep family of oncogenes since human Ga12 was cloned as a transforming oncogene from Ewin's sarcoma expression library (Xu et al., 1994). Analyses of the Ga12Q229L-and Ga13Q226L-transformed ®broblasts have begun to provide clues to their strong growth promoting activities. It has been observed that none of the conventional secondary messenger pathways involving cAMP, Ca2+, or IP3 were altered in these cells (Chan et al., 1993; Xu et al., 1993, 1994; Jiang et al., 1993; Vara Prasad et al., 1994; Voyno-Yasenetskaya et al., 1994). Interestingly, the expression of Ga12Q229L and Ga13Q226L in di€erent cell lines result in the potent activation of JNKs (Vara Prasad et al., 1995; Collins et al., 1996; VoynoYasenetskaya et al., 1996). In this context, it should

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be noted that this is one of the very few instances where the constitutive activation of JNK has an antiapoptotic e€ect. The activation of JNK is mediated by the small GTPases Ras, Rac, CDC42, (Vara Prasad et al., 1995; Collins et al., 1996; Voyno-Yasenetskaya et al., 1996) and Rho (Nagao et al., 1999). Ga12 and Ga13 are also involved in transmitting additional growth-promoting signals (Dhanasekaran and Dermott, 1996). These include the Ras/Racdependent transient activation of ERK (Mitsui et al., 1997), and Rho-dependent activation of focal adhesion complex formation (Buhl et al., 1996; Hooley et al., 1996). While ERK-signaling pathway is not signi®cantly altered in many of these cells, Ga12 weakly stimulates ERK in NIH3T3 cells (Mitsui et al., 1997). Ras-dependent ERK activity as well as Ras/Rac dependent JNK activity were found to be required for NIH3T3 cell progression from G1 to S phase (Mitsui et al., 1997). The synergism among the signaling pathways regulated by Ga12, Raf1, and Rac1 has been shown to be essential for Ga12-mediated transformation of NIH3T3 cells (Zhang et al., 1996; Tokacheva et al., 1997). Ga12 has also been shown to potentiate serum-stimulated arachidonic acid release in NIH3T3 cells (Xu et al., 1993; Dermott et al., 1999). Furthermore, Ga12 has been shown to stimulate the phosphorylation and subsequent activation of several tyrosine kinases such as FAK (Needham and Rozengurt, 1998) and Tec/Bmx kinases (Mao et al., 1998c) and Pyk-2 (Shi et al., 2000). Ga12 activates several other signaling pathways by virtue of its ability to activate the small GTPases Ras, Rac, CDC42, and Rho. Examples of such pathways include the Ras/Rhodependent stimulation of PC-PLC or PLD (Wadsworth et al., 1997; Plonk et al., 1998) and Rac, Rho dependent activation of phosphatidylinositide 4-kinase (PI4K) and phosphatidylinositide 4-phosphate 5-kinase (PIP5K) and PIP5K (Gebauer and Dhanasekaran, unpublished observation). In addition, it has been shown that Ga12 can activate PKC-dependent signaling pathways in other cell types (Dhanasekaran et al., 1994; Wadsworth et al., 1997). Furthermore, speci®c sets of primary response genes appear to be activated in Ga12QL- and Ga13QL -transformed NIH3T3 cells (Fromm et al., 1997; Vara Prasad et al., 1994). Ga12QL has been shown to activate SRFs through a Rhodependent pathway (Fromm et al., 1997). Ga13QL has been shown to activate the transcription of Egr-1 a primary response gene implicated in cell proliferation as well as di€erentiation (Vara Prasad et al., 1994). Similarly, the transcriptional activation of cyclooxygenase-2 or COX-2 pathway by Ga12 and Ga13 pathway can also play a major role in Ga12/13 mediated oncogenic transformation. Recently these additional reinforcements of cell-survival signals should be contributing to the accelerated cell growth seen in cells expressing Ga12QL and Ga13QL. The mechanisms through which Ga12,13 transmits its signals to the di€erent small GTPases are largely unknown. Ga12,13 can stimulate the small GTPases by (1) stimulating speci®c guanine nucleotide exchange

factors (GEFs), (2) competing with guanine nucleotide dissociation inhibitors (GDIs), or (3) inhibiting speci®c GAPs (Dhanasekaran and Dermott, 1996). It has been shown that both Ga12 and Ga13 can physically interact with RGS-motif containing RhoGEF (Kozasa et al., 1998; Hart et al., 1998; Fukuhara et al., 1999). The coupling between Ga12,13 and Rho appear to involve addition mechanisms (Gohla et al., 1998, 1999). It has been observed that signal coupling between Ga13 and Rho appear to involve receptor tyrosine kinases such as EGFR and other non-receptor kinases. In contrast, the coupling between Ga12 and Rho appear to be independent of any tyrosine kinases. Similarly a role for Btk family of kinases in Ga12,13 coupling to Rho has been observed (Mao et al., 1998a,b). The recent ®nding that Shc is involved in transmitting the signals from Ga12 to Ras suggest the mechanism through which Ga12 can stimulate Ras (Collins et al., 1997). The observation that LPA receptors can activate Tiam1, an exchange factor for Rac through a PKCdependent mechanism (Fleming et al., 1997), suggests an interesting possibility that Ga12, which activates PKC-dependent pathway in some cell types, can stimulate Rac and possibly Rac-mediated JNK activity through PKC and Tiam-1. The observation that the activation of JNK by Ga12 is PKC-dependent in HeLa cells ®ts well with this hypothesis (Tsim and Dhanasekaran, unpublished observations). The recent observations that Ga12 can physically interact with a novel RasGAP as well as Bruton's tyrosine kinase and stimulate their activity (Jiang et al., 1998) are of great interest. However, the role of these signaling components in Ga12-stimulated cell proliferation and oncogenic transformation remains to be de®ned. How are the multiple signaling inputs generated from Ga12/13 integrated into a cell proliferation signal? It appears that the signals from Ga12/13 are translated into growth-promoting signals by the small GTPases Ras, Rac, and Rho. It has been reported that Ras, Rho, Rac, and CDC42 play an essential role in cell cycle progression from G1 to S phase (Aktas et al., 1997; Takuwa and Takuwa, 1997; Olson et al., 1995; Hirai et al., 1997). An additional role for Rac in progression from G2 to M phase has also been identi®ed (Moore et al., 1997). Based on these observations, it is likely that Ga12/13 activation of Ras-, Rac-, and Rho-regulated signaling mechanisms accelerate G1/S and G2/M phase cell cycle progression. The observation that Ga12-induced cell proliferation depends on both Ras-regulated ERK and Ras/Racregulated JNK to progress through G1 to S phase provide strong support to this view (Mitsui et al., 1997). Moreover, it is likely Ga12/13-activated Rho plays additional roles. Rho is involved in the regulation of cytoskeletal rearrangements associated with cell division and proliferation (Ridley and Hall, 1992; Takai et al., 1995). Furthermore, Rho has been known to activate speci®c SRFs through a hitherto unidenti®ed mechanism (Hill et al., 1995). Thus activation of Rho, in addition to Ras and Rac may greatly facilitate cell growth in cells expressing Ga12/13. In this context,

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it is signi®cant to note that Ga12-mediated transformation of NIH3T3 cell line is dependent on Rho and Rho-mediated activation of SRFs (Fromm et al., 1997). Thus, Ga12/13 coordinate several critical signaling events through its interactions with the Ras- and Rho-family of GTPases. These include the regulation of di€erent kinase modules as well as the activation of several transcription factors such as SRFs, TCFs, Jun and ATF2. Recently, it has been demonstrated that Ga12QL and Ga13QL stimulates the activation of COX-2 promoter in NIH3T3 cells (Dermott et al., 1999; Slice et al., 1999). In addition, it has been shown that the inhibition of COX-2 using a commercially available COX-2 inhibitor drastically inhibited the DNA synthesis in Ga12QL-NIH3T3 transformants (Dermott, 1999). While this ®nding suggests a major role for eicosanoid pathway in Ga12QL-mediated cell proliferation, it also illustrates an important mechanism underlying the potent transforming activity of Ga12QL. It has been considered that the phospholipase-mediated release of arachidonic acid is the rate-limiting step in the biosynthesis of prostaglandins. However, with the identi®cation of COX-2, it has been realized that the synthesis of prostaglandin is regulated at two distinct loci, namely PLA2 and COX-2 (Reddy and Herschman, 1994). Through the concerted regulation of PLA2 and COX-2, Ga12 ascertains the availability of the substrate (arachidonic acid) as well as the enzyme (COX-2) to maximize the production of prostaglandins and their metabolites. Such a co-stimulation of PLA2 and COX-2 has been proposed to have a causative role in Ras-mediated cellular transformation in human nonsmall cell lung cancer (Heasley et al., 1997). Perhaps, similar ability of Ga12 to regulate more than one locus of the eicosanoid pathway contributes signi®cantly to its aggressive growth promoting activity. Similarly, it has been observed that in addition to the activation of di€erent mitogenic pathways, Ga12QL mediates the inhibition of an apoptotic pathway involving p38MAPK (Dermott and Dhanasekaran, 2001). In

the light of the recent observation that p38MAPK is the primary signaling module involved in the apoptotic pathways activated by serum-deprival (Kummer et al., 1997), this ®nding, for the ®rst time demonstrates the ability of Ga12 to inhibit an apoptotic pathway to promote cell growth. Presumably, the combination of multiple proliferative signals together with a strong anti-apoptotic signal leads to the oncogenic transformation of cells expressing these a-subunits. Perhaps the ability to activate such multiple growth promoting signaling-inputs confers the potent oncogenic property unique to Ga12,13. Considering the potent oncogenic property of Ga12,13, it is likely that activating mutations in Ga12,13 should be highly oncogenic leading to human tumors. However, the role of the activating mutations of Ga12,13 in naturally occurring human tumors remains to be established. Conclusion A closer look at the multitudes of responses regulated by the di€erent transforming G proteins shows the underlying interrelationship between these diverse responses. A comparison of the transforming abilities of the transforming a-subunits clearly indicates that the a-subunits that show the highest degree of such signal integration show the most potent growth promoting activity. Although there are gaps in our understanding of the signaling networks regulated by these G proteins, now that the major signaling components regulated by these a-subunits have been identi®ed, the precise signaling pathways involved in the regulation of cell growth and oncogenesis by these proteins should soon be emerging.

Acknowledgments The work was supported by the National Institutes of Health Grant GM49897 (N Dhanasekaran).

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