Activation state-dependent interaction between Gαq subunits and the Fhit tumor suppressor.

BACKGROUND: The FHIT tumor suppressor gene is arguably the most commonly altered gene in cancer since it is inactivated in about 60% of human tumors. The Fhit protein is a member of the ubiquitous histidine triad proteins which hydrolyze dinucleoside polyphosphates such as Ap3A. Despite the fact that Fhit functions as a tumor suppressor, the pathway through which Fhit inhibits growth of cancer cells remains largely unknown. Phosphorylation by Src tyrosine kinases provides a linkage between Fhit and growth factor signaling. Since many G proteins can regulate cell proliferation through multiple signaling components including Src, we explored the relationship between Gα subunits and Fhit. RESULTS: Several members of the Gαq subfamily (Gα16, Gα14, and Gαq) were found to co-immunoprecipitate with Fhit in their GTP-bound active state in HEK293 cells. The binding of activated Gαq members to Fhit appeared to be direct and was detectable in native DLD-1 colon carcinoma cells. The use of Gα16/z chimeras further enabled the mapping of the Fhit-interacting domain to the α2-ß4 region of Gα16. However, Gαq/Fhit did not affect either Ap3A binding and hydrolysis by Fhit, or the ability of Gαq/16 to regulate downstream effectors including phospholipase Cß, Ras, ERK, STAT3, and IKK. Functional mutants of Fhit including the H96D, Y114F, L25W and L25W/I10W showed comparable abilities to associate with Gαq. Despite the lack of functional regulation of Gq signaling by Fhit, stimulation of Gq-coupled receptors in HEK293 and H1299 cells stably overexpressing Fhit led to reduced cell proliferation, as opposed to an enhanced cell proliferation typically seen with parental cells. CONCLUSIONS: Activated Gαq members interact with Fhit through their α2-ß4 region which may result in enhancement of the growth inhibitory effect of Fhit, thus providing a possible avenue for G protein-coupled receptors to modulate tumor suppression.

Galpha16 activates Ras by forming a complex with tetratricopeptide repeat 1 (TPR1) and Son of Sevenless (SOS).

Many G protein-coupled receptors (GPCRs) are known to modulate cell growth and differentiation by stimulating the extracellular signal-regulated protein kinases (ERKs). In growth factor signaling, ERKs are typically stimulated through an elaborate network of modules consisting of adaptors, protein kinases, and the small GTPase Ras. The mechanism by which G protein signals tap into the ERK signaling pathway has thus far remain elusive. Members of the Gq family of G proteins, in particular Galpha16, have been shown to associate with tetratricopeptide repeat 1 (TPR1), an adaptor protein which preferentially binds to Ras. Here, we examined if TPR1 is indeed the missing link between Galpha16 signaling and Ras activation. Expression of Galpha16QL, a constitutively active mutant of Galpha16, in HEK 293 cells led to the formation of GTP-bound Ras and the subsequent phosphorylation of ERK. Likewise, stimulation of endogenou G16-coupled CCR1 chemokine receptors produced the same responses in human erythroleukemia cells. siRNA-mediated knockdown of TPR1 or expression of a dominant negative mutant of TPR1 effectively abolished the ability of Galpha16QL to induce Ras activation in HEK 293 cells. In contrast, these manipulations had no inhibitory effect on Galpha16QL induced activation of phospholipase Cbeta. Galpha16QL-induced phosphorylations of downstream targets including ERK, signal transducer and activator of transcription 3, and IkappaB kinase were significantly suppressed upon expression of the dominant negative mutant of TPR1. Furthermore, SOS2, a Ras guanine nucleotide exchange factor, was found to form a complex with TPR1 and Galpha16QL. Expression of SOS2 enhanced Galpha16QL-induced Ras activation and its subsequent signaling. Collectively, our results suggest that Galpha16 regulates multiple signaling pathways by activating Ras through its association with TPR1, but TPR1 is not required for Galpha16 to stimulate phospholipase Cbeta.

Galpha16 interacts with Class IA phosphatidylinositol 3-kinases and inhibits Akt signaling.

Phosphatidylinositol 3-kinase (PI3K) mediates receptor tyrosine kinase and G protein coupled receptor (GPCR) signaling by phosphorylating phosphoinositides to elicit various biological responses. Galpha(q) has previously been shown to inhibit class IA PI3K by interacting with the p110alpha subunit. However, it is not known if PI3Ks can associate with other Galpha(q) family members such as Galpha(16). Here, we demonstrated that class IA PI3Ks, p85/p110alpha and p85/p110beta, could form stable complexes with wild type Galpha(16) and its constitutively active mutant (Galpha(16)QL) in HEK293 cells. In contrast, no interaction between Galpha(16) and class IB PI3K was observed. The Galpha(16)/p110alpha signaling complex could be detected in hematopoietic cells that endogenously express Galpha(16). Overexpression of class I PI3Ks did not inhibit Galpha(16)QL-induced IP(3) production and, unlike p63RhoGEF, class IA PI3Ks did not attenuate the binding of PLCbeta(2) to Galpha(16)QL. On the contrary, the function of class IA PI3Ks was suppressed by Galpha(16)QL as revealed by diminished production of PIP(3) as well as inhibition of EGF-induced Akt phosphorylation. Taken together, these results suggest that Galpha(16) can bind to class IA PI3Ks and inhibit the PI3K signaling pathway.

The RhoA-specific guanine nucleotide exchange factor p63RhoGEF binds to activated Galpha(16) and inhibits the canonical phospholipase Cbeta pathway.

Heterotrimeric G proteins regulate diverse physiological processes by modulating the activities of intracellular effectors. Members of the Galpha(q) family link G protein-coupled receptor activation to phospholipase Cbeta (PLCbeta) activity and intracellular calcium signaling cascades. However, they differ markedly in biochemical properties as well as tissue distribution. Recent findings have shown that some of the cellular activities of Galpha(q) family members are independent of PLCbeta activation. A guanine nucleotide exchange factor, p63RhoGEF, has been shown to interact with Galpha(q) proteins and thus provides linkage to RhoA activation. However, it is not known if p63RhoGEF can associate with other Galpha(q) family members such as Galpha(16). In the present study, we employed co-immunoprecipitation studies in HEK293 cells to demonstrate that p63RhoGEF can form a stable complex with the constitutively active mutant of Galpha(16) (Galpha(16)QL). Interestingly, overexpression of p63RhoGEF inhibited Galpha(16)QL-induced IP(3) production in a concentration-dependent manner. The binding of PLCbeta(2) to Galpha(16)QL could be displaced by p63RhoGEF. Similarly, p63RhoGEF inhibited the binding of tetratricopeptide repeat 1 to Galpha(16)QL, leading to a suppression of Galpha(16)QL-induced Ras activation. In the presence of p63RhoGEF, Galpha(16)QL-induced STAT3 phosphorylation was significantly reduced and Galpha(16)QL-mediated SRE transcriptional activation was attenuated. Taken together, these results suggest that p63RhoGEF binds to activated Galpha(16) and inhibits its signaling pathways.

Integration of G protein signals by extracellular signal-regulated protein kinases in SK-N-MC neuroepithelioma cells.

Mammalian cells often receive multiple extracellular stimuli under physiological conditions, and the various signaling inputs have to be integrated for the processing of complex biological responses. G protein-coupled receptors (GPCRs) are critical players in converting extracellular stimuli into intracellular signals. In this report, we examined the integration of different GPCR signals by mitogen-activated protein kinases (MAPKs) using the SK-N-MC human brain neuroepithelioma cells as a neuronal model. Stimulation of the Gi-coupled neuropeptide Y1 and Gq-coupled muscarinic M1 acetylcholine receptors, but not the Gs-coupled dopamine D1 receptor, led to the activation of extracellular signal-regulated kinase (ERK). All three receptors were also capable of stimulating c-Jun NH2-terminal kinases (JNK) and p38 MAPK. The Gi-mediated ERK activation was completely suppressed upon inhibition of Src tyrosine kinases by PP1, while the Gq-induced response was suppressed by both PP1 and the Ca2+ chelator, BAPTA-AM. In contrast, activations of JNK and p38 by Gs-, Gi-, and Gq-coupled receptors were sensitive to PP1 and BAPTA-AM pretreatments. Simultaneous stimulation of Gi- and Gq-coupled receptors resulted in the synergistic activation of ERK, but not JNK or p38 MAPK. The Gi/Gq-induced synergistic ERK activation was PTX-sensitive, and appeared to be a co-operative effect between Ca2+ and Src family tyrosine kinases. Enhanced ERK activation was associated with an increase in CREB phosphorylation, while the JNK and p38-responsive transcription factor ATF-2 was weakly enhanced upon Gi/Gq-induction. This report provides evidence that G protein signals can be integrated at the level of MAPK, resulting in differential effects on ERK, JNK and p38 MAPK in SK-N-MC cells.