Eddy H.T. Wu

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.

Nerve growth factor-induced stimulation of p38 mitogen-activated protein kinase in PC12 cells is partially mediated via Gi/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 Gi/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 Gs, Gq/11 and G12/13 subfamilies in the regulation of Akt in cultured mammalian cells. In human embryonic kidney 293 cells transiently expressing constitutively active mutants of Gα11, Gα14, Gα16, Gα12, or Gα13 (Gα11QL, Gα14QL, Gα16QL, Gα12QL and Gα13QL, 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 Gαs mutant (GαsQL). Additional experiments showed that Gα11QL, Gα14QL, Gα16QL, Gα12QL and Gα13QL, but not GαsQL, 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 Cβ activation and calcium signaling because Gα11QL, Gα14QL and Gα16QL 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 Gα12QL‐ and Gα13QL‐mediated inhibition, suggesting that activated Gα12 and Gα13 inhibit Akt signaling via RhoA. Collectively, this study demonstrated the inhibitory effect of activated Gα11, Gα14, Gα16, Gα12 and Gα13 on pro‐survival Akt signaling.

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., Gi/o) as well as inhibit (e.g., Gq) 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.

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

The Gi‐coupled M4 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 M4 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 Gi‐coupled M4 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 M4 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 M4 mAChR and TrkA, respectively.