N. Dhanasekaran

Regulation of cell proliferation by G proteins.

G Proteins provide signal transduction mechanisms to seven transmembrane receptors. Recent studies have indicated that the α-subunits as well as the βγ-subunits of these proteins regulate several critical signaling pathways involved in cell proliferation, differentiation and apoptosis. Of the 17 α-subunits that have been cloned, at least ten of them have been shown to couple mitogenic signaling in fibroblast cells. Activating mutations in Gαs, Gαi2, and Gα12 have been correlated with different types of tumors. In addition, the ability of the βγ-subunits to activate mitogenic pathways in different cell-types has been defined. The present review briefly summarizes the diverse and novel signaling pathways regulated by the α- as well as the βγ-subunits of G proteins in regulating cell proliferation.

Signaling by the G12 class of G proteins

The G12 class of G proteins are defined by the alpha-subunits of mammalian G12 and G13. Biochemical and mutational characterization of G alpha 12/13 have identified several novel signaling pathways regulated by these alpha-subunits. Studies with the constitutively activated mutants of G alpha 12 and G alpha 13 have indicated that they stimulate mitogenic signaling pathways leading to the oncogenic transformation of fibroblast cell lines. Recent analyses have indicated that G alpha 12 and G alpha 13 regulate cytoplasmic as well as nuclear signaling events such as activation of the Jun N-terminal kinase signaling module, Na+/H+ exchangers, focal adhesion assemblies, and transcriptional activation of specific primary response genes. The emerging view suggests that these signaling events represent an integrated response regulated by G12 and G13. This review discusses the diverse signaling responses regulated by G12 and G13, and the interrelationship of these responses.

Transforming G proteins

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 α, β, and γ subunits. The β- and γ-subunits are tightly associated. The receptors activated by the appropriate ‘signal’, interact catalytically with specific G-proteins to mediate guanine nucleotide exchange at the GDP/GTP binding site of the G-protein α-subunits, thus displacing the bound GDP for GTP. The GTP bound form of the g-protein α-subunit and in some cases the free βγ-subunits initiate cellular response by altering the activity of specific effector molecules. Recent studies have indicated that the asyncronous activation of these proteins can lead to the oncogenic transformation of different cell types. The mechanism by which G-proteins regulate the various cell functions appear to involve a complex net-working between different signaling pathways. This review summarizes the signaling mechanisms involved in the regulation of cell proliferation by these transforming G proteins.

JLP: A scaffolding protein that tethers JNK/p38MAPK signaling modules and transcription factors

Extracellular signals are transduced into cells through mitogen-activated protein kinases (MAPKs), which are activated by their upstream kinases. Recently, families of scaffolding proteins have been identified to tether specific combinations of these kinases along specific signaling pathways. Here we describe a protein, JLP (c-Jun NH2-terminal kinase-associated leucine zipper protein), which acts as a scaffolding protein to bring together Max and c-Myc along with JNK (c-Jun NH2-terminal kinase) and p38MAPK, as well as their upstream kinases MKK4 (MAPK kinase 4) and MEKK3 (MAPK kinase kinase 3). Thus, JLP defines a family of scaffolding proteins that bring MAPKs and their target transcription factors together for the execution of specific signaling pathways.

Apoptosis of ovarian granulosa cells: correlation with the reduced activity of ERK-signaling module.

Apoptosis of the ovarian granulosa cells plays a crucial role in the determination of the number of follicles destined to ovulate in each reproductive cycle. While the activation of specific apoptotic pathway or the inactivation of cell survival pathway can initiate apoptosis, the signaling mechanism(s) involved in initiating the onset of apoptosis in granulosa cells is not fully understood. In the present study, using granulosa cells derived from eCG‐primed immature rats, we investigated the temporal signaling events involved in the onset of apoptosis in the granulosa cells. The administration of 15 IU of eCG to 21‐day‐old immature female rats stimulate the growth and development of ovarian follicles until 72 h, after which the granulosa cells of the ovarian follicles undergo apoptosis due to the waning levels of tropic hormonal support. An analysis of the signaling events leading to apoptosis indicates that the DNA fragmentation can be seen in these cells from 96 h. A small increase in the levels of the pro‐apoptotic factor Bax can be seen from 96 h while an increase in the activity of JNK can be seen from 108 h onwards. By contrast, a reduction in ERK signaling can be seen by 48 h. Similar reduction in Raf‐1 kinase activity can be discerned from 48 h onwards. A concomitant decrease in the phosphorylated form of Bad can also be detected. These findings taken together, suggest that the loss of tropic hormone support is translated into the attenuation of Raf‐1‐MEK‐ERK signaling pathway and this reduction along with a reduction in the levels of phosphorylated form of Bad triggers the onset of apoptosis in the ovarian granulosa cells. J. Cell. Biochem. 75:547–554, 1999.

Mitogenic signaling by lysophosphatidic acid (LPA) involves Gα12

Lysophosphatidic acid (LPA), a major G protein coupled receptor (GPCR)-activating ligand present in serum, elicits growth factor like responses by stimulating specific GPCRs coupled to heterotrimeric G proteins such as Gi, Gq, and G12/13. Previous studies have shown that the overexpression of wild-type Gα12 (Gα12WT) results in the oncogenic transformation of NIH3T3 cells (Gα12WT-NIH3T3) in a serum-dependent manner. Based on the potent growth-stimulating activity of LPA and the presence of LPA and LPA-like molecules in the serum, we hypothesized that the serum-dependent neoplastic transformation of Gα12WT-NIH3T3 cells was mediated by the stimulation of LPA-receptors (LPARs) by LPA in the serum. In the present study, using guanine nucleotide exchange assay and GST-TPR binding assay, we show that the treatment of Gα12WT-NIH3T3 with 2 μM LPA leads to the activation of Gα12. Stimulation of these cells with LPA promotes JNK-activation, a critical component of Gα12-response and cell proliferation. We also show that LPA can substitute for serum in stimulating JNK-activity, DNA synthesis, and proliferation of Gα12WT-NIH3T3 cells. LPA-mediated proliferative response in NIH3T3 cells involves Gα12, but not the closely related Gα13. Pretreatment of Gα12WT-NIH3T3 cells with suramin (100 μM), a receptor-uncoupling agent, inhibited LPA-stimulated proliferation of these cells by 55% demonstrating the signal coupling between cell surface LPAR and Gα12 in the neoplastic proliferation of NIH3T3 cells. As LPA and LPAR mediated mitogenic pathways have been shown to play a major role in tumor genesis and progression, a mechanistic understanding of the signal coupling between LPAR, Gα12, and the downstream effectors is likely to unravel additional targets for novel cancer chemotherapies.

Ras-dependent Signaling by the GTPase-deficient Mutant of Gα12

Gα12 and Gα13regulate diverse responses through the small GTPases Ras, CDC42, Rac, and Rho. Whereas they activate similar responses in many different cell types, they also activate more specific and critical signaling pathways in other cell types. In COS cells, in which both Gα12 and Gα13 stimulate Na+/H+ exchange, they do so by activating different signaling pathways. Here we report that the differential recruitment of specific small GTPases by Gα12 and Gα13 defines the molecular basis for their functional differences. We have observed that the stimulation of Na+/H+ exchange by the GTPase-deficient mutant of Gα12 (Gα12QL) requires a functional Ras and is independent of Rac/CDC42 and Jun kinase signaling module. By contrast, the stimulation of Na+/H+ exchange by Gα13QL requires a functional Rac/CDC42 and the Jun kinase signaling module. Our results also indicate that Gα12QL-Ras stimulation of Na+/H+ exchange involves a D609-sensitive phospholipase and protein kinase C. These studies, for the first time, describe a novel Gα12-specific signaling pathway involving Ras, phosphatidylcholine hydrolysis, and protein kinase C in the regulation of Na+/H+ exchange.

Oncogenic mutant of Gα12 stimulates cell proliferation through cycloxygenase-2 signaling pathway

Expression of the GTPase-deficient, activated mutant α-subunit of the heterotrimeric G protein G12 (Gα12QL) leads to the neoplastic transformation of fibroblast cell lines. The mitogenic pathway regulated by Gα12QL includes an extensive signaling network involving several small GTPases and various kinases. In addition, Gα12QL has been shown to potentiate the serum-induced phospholipase-A2 activity in NIH3T3 cells. In the present study, we demonstrate that cycloxygenase-2 (COX-2) pathway is involved in the mitogenic pathway activated by Gα12QL. Expression of Gα12QL and not Gα13QL, stimulates the serum-induced release of arachidonic acid in NIH3T3 cells. Furthermore, expression of Gα12QL or the stimulation of wild-type Gα12 induces the expression of COX-2. Our results also indicate that the COX-2 inhibitor acutely disrupts the DNA-synthesis stimulated by Gα12QL in NIH3T3 cells. These studies, for the first time, identify the crucial role of COX-2 in Gα12-mediated regulation of cell proliferation and suggest a role for prostaglandin-derived autocrine loop in Gα12-mediated signaling pathways.

G Protein Subunits and Cell Proliferation

Heterotrimeric, guanine nucleotide binding proteins, known as G proteins, provide signaling mechanisms for the serpentine family of receptors. Recent studies indicate that the α- as well as the βγ-subunits of the G proteins are involved in the regulation of several cellular responses. Some of these responses proved to be critical for the regulation of cell growth and differentiation. Studies using the constitutively activated mutants of the Gα subunits and the overexpression of Gβγ subunits have indicated that these different subunits regulate cell proliferation through diverse signaling pathways involving distinct low molecular weight GTPases and specific protein kinases. The integrated networking between these different pathways finally defines the coordinated regulation of cell proliferation. This review briefly summarizes our present understanding of the different signaling mechanisms involved in the regulation of cell proliferation by the different Gα and Gβγ subunits.

Differential regulation of Jun N-terminal kinase and p38MAP kinase by Gα12

Based on the findings that the overexpression of the wild-type Gα12 (Gα12WT) result in the oncogenic transformation of NIH3T3 cells in a serum-dependent manner, a model system has been established in which the mitogenic and subsequent cell transformation pathways activated by Gα12 can be turned on or off by the addition or removal of serum. Using this model system, our previous studies have shown that the stimulation of Gα12WT or the expression of an activated mutant of Gα12 (Gα12QL) leads to increased cell proliferation and subsequent oncogenic transformation of NIH3T3 cells, as well as persistent activation of Jun N-terminal kinases (JNKs). In the present studies, we show that the stimulation of Gα12WT or the expression of Gα12QL results in a potent inhibition of p38MAPK, and that the mechanism by which Gα12 inhibits p38MAPK activity involves the dual specificity kinases upstream of p38MAPK. The results indicate that Gα12 attenuates the activation of MKK3 and MKK4, which are known to stimulate only p38MAPK or p38MAPK and JNK, respectively. The results also suggest that Gα12 activates JNKs specifically through the stimulation of the JNK-specific upstream kinase MKK7. These findings demonstrate for the first time that Gα12 differentially regulates JNK and p38MAPK by specifically activating MKK7, while inhibiting MKK3 and MKK4 in NIH3T3 cells. Since the stimulation of p38MAPK is often associated with apoptotic responses, our findings suggest that Gα12 stimulates cell proliferation and neoplastic transformation of NIH3T3 cells by attenuating p38MAPK-associated apoptotic responses, while activating the mitogenic responses through the stimulation of ERK- and JNK-mediated signaling pathways.