Regulator of G protein signaling 19 suppresses Ras-induced neoplastic transformation and tumorigenesis.

Regulator of G protein signaling 19 (RGS19) has recently been shown to inhibit Ras activation by upregulating the tumor metastasis suppressor Nm23. Here, we have examined the effect of RGS19 on Ras-induced oncogenesis. Coexpression of RGS19, but not RGS20, in NIH3T3 cells effectively suppressed neoplastic transformation and tumorigenesis induced by the oncogenic Ras(GV) mutant. In non-small cell lung carcinoma H1299 cells that harbor Ras mutations, shRNA-mediated knockdown of RGS19 facilitated tumorigenesis with the early appearance of large tumors in nude mice assays. Collectively, these results suggest that expression of RGS19 can suppress the oncogenic actions of Ras.

RGS19 inhibits Ras signaling through Nm23H1/2-mediated phosphorylation of the kinase suppressor of Ras.

Besides serving as signal terminators for G protein pathways, several regulators of G protein signaling (RGS) can also modulate cell proliferation. RGS19 has previously been shown to enhance Akt signaling despite impaired Ras signaling. The present study examines the mechanism by which RGS19 inhibits Ras signaling. In HEK293 cells stably expressing RGS19, serum-induced Ras activation and phosphorylations of Raf/MEK/ERK were significantly inhibited, while cells expressing RGS2, 4, 7, 8, 10, or 20 did not exhibit this inhibitory phenotype. Conversely, siRNA-mediated knockdown of RGS19 enabled partial recovery of serum-induced ERK phosphorylation. Interestingly, two isoforms of the tumor metastasis suppressor Nm23 (H1 and H2) were upregulated in 293/RGS19 cells. As a nucleoside diphosphate kinase, Nm23H1 can phosphorylate the kinase suppressor of Ras (KSR). Elevated levels of phosphorylated KSR were indeed detected in the nuclear fractions of 293/RGS19 cells. Co-immunoprecipitation assays revealed that Nm23H1/2 can form complexes with RGS19, Ras, or KSR. siRNA-mediated knockdown of Nm23H1/2 allowed 293/RGS19 cells to partially recover their ERK responses to serum treatment, while overexpression of Nm23H1/2 in HEK293 cells suppressed the serum-induced ERK response. This study demonstrates that expression of RGS19 can suppress Ras-mediated signaling via upregulation of Nm23.

Elevated expression of RGS19 impairs the responsiveness of stress-activated protein kinases to serum.

Regulators of G protein signaling (RGS proteins) serve as GTPase activating proteins for the signal transducing Gα subunits. RGS19, also known as Gα-interacting protein (GAIP), has been shown to subserve other functions such as the regulation of macroautophagy and growth factor signaling. We have recently demonstrated that the expression of RGS19 in human embryonic kidney (HEK) 293 cells resulted in the disruption of serum-induced mitogenic response along the classical Ras/Raf/MEK/ERK pathway. Here, we further examined the effect of RGS19 expression on the stress-activated protein kinases (SAPKs). Both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) became non-responsive to serum in 293/RGS19 cells, yet the two SAPKs responded to UV irradiation or osmotic stress induced by sorbitol. Kinases upstream of JNK and p38 MAPK, including MKK3/6, MKK4, and MLK3, also failed to respond to serum stimulation in 293/RGS19 cells. Serum-induced activation of the small GTPases Rac1 and Cdc42 was similarly suppressed in these cells. Our results indicate that elevated expression of RGS19 can severely disrupt the regulation of MAPKs by small GTPases.

RGS19 stimulates cell proliferation by deregulating cell cycle control and enhancing Akt signaling.

RGS19 is a regulator of G protein signaling which is upregulated in ovarian cancers and its overexpression promotes cell proliferation in several mammalian cell types. Here we showed that cyclin D1/3 and Cdk6 were upregulated in HEK293 cells overexpressing RGS19, while INK4A and INK4B were reduced. Moreover, RGS19 augmented serum-stimulated PTEN/PDK/Akt and Rb phosphorylations in 293/RGS19 and Caco2/RGS19 cells. These changes were reversed upon the knockdown of RGS19. Consistent with an elevated Akt activity, increased levels of phosphorylated Bad and c-Raf and a diminished expression of TSC2 were detected, thus demonstrating that RGS19 can deregulate cell proliferation via multiple pathways.

RGS19 enhances cell proliferation through its C-terminal PDZ motif.

Regulator of G protein signaling 19 (RGS19), also known as Galpha-interacting protein (GAIP), is a GTPase activating protein (GAP) for Galpha(i) subunits. Apart from its GAP function, RGS19 has been implicated in growth factor signaling through binding to GAIP-interacting protein C-terminus (GIPC) via its C-terminal PDZ-binding motif. To gain additional insight on its function, we have stably expressed RGS19 in a number of mammalian cell lines and examined its effect on cell proliferation. Interestingly, overexpression of RGS19 stimulated the growth of HEK293, PC12, Caco2, and NIH3T3 cells. This growth promoting effect was not shared by other RGS proteins including RGS4, RGS10 and RGS20. Despite its ability to stimulate cell proliferation, RGS19 failed to induce neoplastic transformation in NIH3T3 cells as determined by focus formation and soft-agar assays, and it did not induce tumor growth in athymic nude mice. Deletion mutants of RGS19 lacking the PDZ-binding motif failed to complex with GIPC and did not exhibit any growth promoting effect. Overexpression of GIPC alone in HEK293 cells stimulated cell proliferation whereas its knockdown in H1299 non-small cell lung carcinomas suppressed cell proliferation. This study demonstrates that RGS19, in addition to acting as a GAP, is able to stimulate cell proliferation in a GIPC-dependent manner.

Multiple Gi proteins participate in nerve growth factor-induced activation of c-Jun N-terminal kinases in PC12 cells.

Nerve growth factor (NGF)-mediated activation of mitogen-activated protein kinases (MAPK) is critical for differentiation and apoptosis of PC12 cells. Since NGF employs stress-activated c-Jun N-terminal kinase (JNK) to regulate both programmed cell death and neurite outgrowth of PC12 cells, we examined NGF-regulated JNK activity and the role of G(i/o) proteins. Induction of JNK phosphorylation by NGF occurred in a time- and dose-dependent manner and was partially inhibited by pertussis toxin (PTX). To discern the participation of various signaling intermediates, PC12 cells were treated with specific inhibitors prior to NGF challenge. NGF-elevated JNK activity was abolished by inhibitors of JNK, p38 MAPK, Src, JAK3 and MEK1/2. NGF-dependent JNK phosphorylation became insensitive to PTX treatment upon transient expressions of Galpha(z) or the PTX-resistant mutants of Galpha(i1-3) and Galpha(oA). Collectively, these studies indicate that NGF-dependent JNK activity may be mediated via G(i1-3) proteins, JAK3, Src, p38 MAPK and the MEK/ERK cascade.

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.

Opioid receptor-like (ORL1) receptor utilizes both G(oA) and G(oB) for signal transduction.

The ORL1 receptors stably expressed in HEK 293 cells can utilize PTX-resistant mutants of Galpha(oA/B) to inhibit adenylyl cyclase (AC) and stimulate extracellular signal-regulated protein kinases (ERKs). However, development of AC superactivation and loss of ERK1/2 responsiveness induced by chronic activation of the ORL1 receptors remained PTX-sensitive.

Molecular basis of opioid dependence: role of signal regulation by G-proteins.

1. Morphine and opiate narcotics are potent analgesics that have a high propensity to induce tolerance and physical dependence following their repeated administration. 2. The molecular basis of opiate dependence has not been completely elucidated, although the participation of opioid receptors is a prerequisite. Cellular dependence on opioids is believed to result from the chronic stimulation of opioid-regulated signalling networks. 3. As G-protein-coupled receptors, the opioid receptors must rely on heterotrimeric G-proteins for signal transduction. Recent advances in our understanding of G-protein signalling have unveiled novel signalling molecules and mechanisms, some of which may be intricately involved in the manifestation of opiate dependence. 4. In the present review, we will attempt to trace chronic opioid signals along elaborate G-protein-regulated pathways.