Regulation of mTOR and p70 S6 kinase by the muscarinic M4 receptor in PC12 cells.

The G(i)-coupled M(4) muscarinic acetylcholine receptor (mAChR) has recently been shown to stimulate the survival of PC12 cells through the PI3K/Akt/tuberin pathway. Since mTOR and p70S6K are critical components in activating translation which lie downstream of tuberin, we examined the ability of M(4) mAChR to regulate these targets in PC12 cells. Carbachol (CCh) dose-dependently stimulated both mTOR and p70S6K phosphorylations and these responses were abolished by pertussis toxin pretreatment, indicating the involvement of the G(i)-coupled M(4) mAChR. Phosphorylations of both mTOR and p70S6K were effectively blocked upon inhibition of PI3K by wortmannin. As compared to similar responses elicited by the nerve growth factor (NGF), the M(4) mAChR-induced activation of Akt/tuberin/mTOR/p70S6K occurred in a relatively transient manner. Although inhibition of protein phosphatase 2A by okadaic acid augmented the transient effects of CCh on Akt/tuberin phosphorylations, it failed to significantly prolong these responses. The total protein level of PTEN (tumor suppressor gene phosphatase and tensin homologue deleted on chromosome ten) was attenuated upon NGF, but not CCh treatment. This indicates that downregulation of PTEN may help to sustain the phosphorylation of Akt/tuberin by NGF. Collectively, these findings suggest that PP2A and PTEN may be involved in fine tuning the regulation of Akt/tuberin/mTOR/p70S6K in PC12 cells by M(4) mAChR and TrkA, respectively.

Nerve growth factor-induced stimulation of p38 mitogen-activated protein kinase in PC12 cells is partially mediated via G(i/o) proteins.

Differentiation of PC12 cells by nerve growth factor (NGF) requires the activation of various mitogen-activated protein kinases (MAPKs) including p38 MAPK. Accumulating evidence has suggested cross-talk regulation of NGF-induced responses by G protein-coupled receptors, thus we examined whether NGF utilizes G(i/o) proteins to regulate p38 MAPK in PC12 cells. Induction of p38 MAPK phosphorylation by NGF occurred in a time- and dose-dependent manner and was partially inhibited by pertussis toxin (PTX). NGF-dependent p38 MAPK phosphorylation became insensitive to PTX treatment upon transient expressions of Galpha(z) or the PTX-resistant mutants of Galpha(i2) and Galpha(oA). Moreover, Galpha(i2) was co-immunoprecipitated with the TrkA receptor from PC12 cell lysates. To discern the participation of various signaling intermediates, PC12 cells were treated with a panel of specific inhibitors prior to the NGF challenge. NGF-induced p38 MAPK phosphorylation was abolished by inhibitors of Src (PP1, PP2, and SU6656) and MEK1/2 (U0126). Inhibition of the p38 MAPK pathway also suppressed NGF-induced PC12 cell differentiation. In contrast, inhibitors of JAK2, phospholipase C, protein kinase C and Ca(2+)/calmodulin-dependent kinase II did not affect the ability of NGF to activate p38 MAPK. Collectively, these studies indicate that NGF-dependent p38 MAPK activity may be mediated via G(i2) protein, Src, and the MEK/ERK cascade.

Constitutively active alpha subunits of G(q/11) and G(12/13) families inhibit activation of the pro-survival Akt signaling cascade.

Accumulating evidence indicates that G protein signaling plays an active role in the regulation of cell survival. Our previous study demonstrated the regulatory effects of G(i/o) proteins in nerve growth factor-induced activation of pro-survival Akt kinase. In the present study we explored the role of various members of the G(s), G(q/11) and G(12/13) subfamilies in the regulation of Akt in cultured mammalian cells. In human embryonic kidney 293 cells transiently expressing constitutively active mutants of G alpha11, G alpha14, G alpha16, G alpha12, or G alpha13 (G alpha11QL, G alpha14QL, G alpha16QL, G alpha12QL and G alpha13QL, respectively), basal phosphorylation of Akt was attenuated, as revealed by western blotting analysis using a phosphospecific anti-Akt immunoglobulin. In contrast, basal Akt phosphorylation was unaffected by the overexpression of a constitutively active G alpha(s) mutant (G alpha(s)QL). Additional experiments showed that G alpha11QL, G alpha14QL, G alpha16QL, G alpha12QL and G alpha13QL, but not G alpha(s)QL, attenuated phosphorylation of the Akt-regulated translation regulator tuberin. Moreover, they were able to inhibit the epidermal growth factor-induced Akt activation and tuberin phosphorylation. The inhibitory mechanism of Gq family members was independent of phospholipase Cbeta activation and calcium signaling because G alpha11QL, G alpha14QL and G alpha16QL remained capable of inhibiting epidermal growth factor-induced Akt activation in cells pretreated with U73122 and the intracellular calcium chelator, BAPTA/AM. Finally, overexpression of the dominant negative mutant of RhoA blocked G alpha12QL- and G alpha13QL-mediated inhibition, suggesting that activated G alpha12 and G alpha13 inhibit Akt signaling via RhoA. Collectively, this study demonstrated the inhibitory effect of activated G alpha11, G alpha14, G alpha16, G alpha12 and G alpha13 on pro-survival Akt signaling.

Activation of muscarinic M4 receptor augments NGF-induced pro-survival Akt signaling in PC12 cells.

Survival or death of neurons during development is mediated by the integration of a diverse array of signal transduction cascades that are controlled by the availability and acquisition of neurotrophic factors and agonists acting at G protein-coupled receptors (GPCRs). Recent studies have demonstrated that GPCRs can modulate signals elicited by receptor tyrosine kinases (RTK) and vice versa. Here, we examined the activity of pro-survival Akt kinase, in response to stimulation by muscarinic acetylcholine receptors (mAChRs) and co-activation with the nerve growth factor (NGF) receptor in PC12 cells endogenously expressing Gi-coupled M4 mAChR and Gq-coupled M1 and M5 mAChRs. Western blotting analysis using a phosphospecific anti-Akt antibody revealed a dose- and time-dependent increase in Akt phosphorylation in cells stimulated with mAChR specific agonist carbachol (CCh). Co-stimulation with CCh and NGF resulted in augmentation of Akt activity in a pertussis toxin (PTX)-sensitive manner, suggesting that M4 mAChR, but not M1 and M5 mAChRs, was associated with this synergistic Akt activation. The use of transducin as a Gbetagamma scavenger indicated that Gbetagamma subunits rather than Galphai/o acted as the signal transducer. Additional experiments showed that CCh treatment augmented NGF-induced phosphorylation and degradation of the Akt-regulated translation regulator tuberin. This augmentation was also inhibited by PTX pre-treatment or overexpression of transducin. Finally, co-stimulation of PC12 cells with CCh and NGF resulted in enhancement of cell survival. This is the first study that demonstrates the augmentation effect between M4 mAChR and NGF receptor, and the regulatory role of mAChR on tuberin.

Tuberin: a stimulus-regulated tumor suppressor protein controlled by a diverse array of receptor tyrosine kinases and G protein-coupled receptors.

Tuberin, a tumor suppressor protein, is involved in various cellular functions including survival, proliferation, and growth. It has emerged as an important effector regulated by receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). Regulation of tuberin by RTKs and GPCRs is highly complex and dependent on the type of receptors and their associated signaling molecules. Apart from Akt, the first kinase recognized to phosphorylate and inactivate tuberin upon growth factor stimulation, an increasing number of kinases upstream of tuberin have been identified. Furthermore, recruitment of different scaffolding adaptor components to the activated receptors appears to play an important role in the regulation of tuberin activity. More recently, the differential regulation of tuberin by various G protein family members have also been intensively studied, it appears that G proteins can both facilitate (e.g., G(i/o)) as well as inhibit (e.g., G(q)) tuberin phosphorylation. In the present review, we attempt to summarize our emerging understandings of the roles of RTKs, GPCRs, and their cross-talk on the regulation of tuberin.

Activation of delta-, kappa-, and mu-opioid receptors induces phosphorylation of tuberin in transfected HEK 293 cells and native cells.

A number of G protein-coupled receptors have been shown to stimulate tuberin phosphorylation, which is critical for the regulation of translation and is apparently involved in neurotrophin-promoted survival of serum-deprived cells. Here, in HEK 293 cells transiently expressing the delta-, kappa-, or mu-opioid receptors, Western blotting analysis using a phosphospecific anti-tuberin antibody revealed a dose- and time-dependent increase in tuberin phosphorylation upon stimulation by specific opioid agonists. In NG108-15, PC12, and SH-SY5Y cells that endogenously express delta-, kappa-, and mu-opioid receptors, respectively, specific opioid agonists also stimulated tuberin phosphorylation in a dose- and time-dependent manner. Pretreatment of cells with pertussis toxin or PI3K inhibitor wortmannin blocked the opioid-stimulated tuberin phosphorylation, implicating the possible involvement of the G(i/o) proteins and the phosphatidylinositol-3 kinase/Akt pathway in opioid-induced tuberin phosphorylation. This is the first study that demonstrates the regulatory role of opioid receptors on tuberin.

Pertussis toxin-sensitive Gi/o proteins are involved in nerve growth factor-induced pro-survival Akt signaling cascade in PC12 cells.

In Galpha(z)-deficient mice, survival of sympathetic neurons is significantly attenuated in the presence of pertussis toxin (PTX). This suggests that G(i/o) proteins may have distinct roles in neuronal survival. Here, we investigated the possible involvement of G(i/o) proteins in nerve growth factor (NGF)-induced pro-survival phosphatidylinositol-3-kinase (PI3K)/Akt signaling in rat pheochromocytoma PC12 cells. Treatment of PC12 cells with NGF increased the Akt phosphorylation level in a time- and dose-dependent manner. The NGF-dependent Akt activation was partially attenuated by PTX or overexpression of regulators of G protein signaling Z1 (RGSZ1) and Galpha-interacting protein (GAIP)), indicating the participation of G(i/o) proteins. In contrast, epidermal growth factor (EGF)-mediated Akt phosphorylation was unaffected by PTX or RGSZ1 and GAIP. Expression of PTX-resistant mutants of Galpha(i1), Galpha(i3), Galpha(oA), and Galpha(oB), but not Galpha(i2), abolished the inhibitory effect of PTX on NGF-induced Akt activation. The use of transducin as a Gbetagamma scavenger further revealed that Gbetagamma subunits rather than Galpha(i/o) acted as the signal transducer. The activation profiles of Akt-regulated downstream effectors such as Bad, IKK, and nuclear factor-kappaB (NFkappaB) were also examined. NGF-stimulated phosphorylation of Bad and IKK and transcriptional activity of NFkappaB were indeed sensitive to treatments with PTX. This is the first study that demonstrates the involvement of G(i/o) proteins in NGF-induced Akt signaling.

Involvement of G i/o proteins in nerve growth factor-stimulated phosphorylation and degradation of tuberin in PC-12 cells and cortical neurons.

Tuberin is a critical translation regulator whose role in nerve growth factor (NGF)-promoted neuronal survival has not been documented. In the present study, we examined the ability of NGF to regulate tuberin in PC-12 cells and primary cortical neurons. Incubation of serum-deprived cells with NGF stimulated tuberin phosphorylation and induced proteosome-mediated tuberin degradation. Inhibition of the phosphatidylinositol-3-kinase (PI3K) by wortmannin or overexpression of the kinase dead Akt mutant completely blocked the NGF-induced tuberin phosphorylation and degradation. It is interesting that the NGF-induced tuberin phosphorylation was partially blocked by pertussis toxin or overexpression of regulators of G protein signaling (regulator of G protein signaling Z1 and Galpha-interacting protein), suggesting the participation of G(i/o) proteins. The use of transducin as a Gbetagamma scavenger indicated that Gbetagamma subunits rather than Galpha(i/o) acted as the signal transducer. Epidermal growth factor can similarly induce tuberin phosphorylation and degradation via a PI3K/Akt pathway in PC-12 cells, but these responses were insensitive to pertussis toxin treatment. Treatment of PC-12 cells with a specific agonist to the G(i)-coupled alpha(2)-adrenoceptor also stimulated tuberin phosphorylation transiently, further demonstrating the involvement of G(i/o) signaling in tuberin regulation in PC-12 cells. Finally, overexpression of nonphosphorylable tuberin attenuated NGF-promoted survival of PC-12 cells, suggesting that the phosphorylation and degradation of tuberin are important for NGF-promoted cell survival. Together, this study demonstrates the regulatory effect of NGF and G(i/o) signaling on tuberin.

Regulation of STAT3 activity by G16-coupled receptors.

A number of G protein-coupled receptors (GPCRs) have been shown to stimulate signal transducers and activators of transcription (STAT) activities while STAT3 activation by G alpha(o) can lead to neoplastic transformation in fibroblasts. In the present study we examined the ability of GPCRs to activate STAT3 via G alpha(16), a G alpha subunit which is primarily expressed in hematopoietic cells. In HEK 293 cells expressing a STAT3-driven luciferase reporter, the G alpha(16)-coupled ORL(1) and fMLP receptors stimulated luciferase activity upon activation by their agonists. Agonist-induced STAT3 activity required coexpression of G alpha(16) and was resistant to PTX treatment. Upon activation of the ORL(1) and fMLP receptors, phosphorylation of STAT3 at Tyr(705) was detected by immunoblot analysis. Additional experiments indicated that GPCR-mediated STAT3 activation was dependent on JAK and Raf1 signaling, but did not require phosphatidylinositol 3-kinase. This is the first study that demonstrates the stimulatory effect of ORL(1) and fMLP receptors on STAT3 activity.