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

Abstract

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.

Introduction

The small GTPase Ras and its downstream kinases (Raf/MEK/ERK) constitute a major signaling pathway by which growth factors stimulate cell proliferation [1]. The Ras pathway is also important for cancer progression and maintenance and approximately 20–30% of human malignancies are known to express a mutated Ras. The importance of the Ras signaling pathway is further exemplified by the wide spectrum of biological processes that are regulated by the phosphorylation and activation of extracellular signal-regulated protein kinase (ERK). Phosphorylated ERK regulates numerous transcription factors, thereby modulating cell proliferation, survival, and differentiation, as well as controlling other cellular processes such as cell migration [2]. In recent years, it has become apparent that activation of the Ras signaling cascade is often required for G protein-mediated stimulation of transcription factors [3], [4], and many G protein-coupled receptors (GPCRs) possess the ability to stimulate ERK and induce mitogenesis [5].

Despite substantial evidence indicating that GPCRs can stimulate Ras signaling, there is no consensus as to how the signals are relayed. Multiple mechanisms and pathways are available for GPCRs to activate the Ras signaling cascade, including interaction with adapter proteins [6] and clathrin-mediated endocytosis [7]. Each of these processes can be modulated and fine-tuned through elaborate regulatory controls. Given the increasing involvement of GPCRs in the regulation of cell growth and proliferation [5], one might expect that Ras-induced tumorigenesis could be modulated by G protein signals. In this regard, the family of regulators of G protein signaling (RGS proteins) may act as tumor suppressors by rapidly terminating growth signals that originate from GPCRs, or they may facilitate tumorigenesis by removing inhibitory GPCR signals. Indeed, one of the emerging non-canonical functions of RGS proteins is the control of cell growth and proliferation [8]. Changes in the transcript levels of at least 14 RGS proteins have now been linked to the initiation and progression of a multitude of cancers [9]. However, with a few exceptions such as RGS17 [10], the precise mechanisms by which RGS proteins exert their effects on cell proliferation remain largely unknown.

RGS19 (also known as Gα-interacting protein, GAIP) was first identified through screening of Gα_i3 interacting proteins by the yeast two-hybrid assay [11]. Together with RGS17, RGS20 and Ret-RGS, it belongs to the RZ/A subfamily of RGS proteins. Apart from its ability to act as a GTPase activating protein (GAP) for Gα subunits, RGS19 has been shown to regulate macroautophagy, protein degradation, cell proliferation and differentiation. RGS19 transcription is up-regulated in some primary malignant ovarian cancers [12], and overexpression of RGS19 can significantly reduce lysophosphatidic acid (LPA; a key mediator for ovarian cancer initiation and progression) signaling in CAOV-3 ovarian cancer cells [13]. Linkage of RGS19 to cell growth and proliferation may occur via its interaction with GAIP-interacting protein C-terminus (GIPC) [14], an adapter protein which associates with growth factor receptors [15] and is up-regulated in primary gastric cancer [16]. The ability of RGS19 to enhance cell proliferation in HEK293 cells appears to depend on GIPC because the growth promoting effect is not seen with a RSG19 truncation mutant that fails to bind GIPC [17]. Nevertheless, despite its ability to stimulate cell proliferation, RGS19 does not induce neoplastic transformation in NIH3T3 fibroblasts [17].

Given that the expression of RGS19 is apparently associated with tumorigenesis [9] and that its expression can be upregulated by vascular endothelial growth factor-B [18], it is important to understand how changes in the protein level of RGS19 affect cell proliferation. Surprisingly, expression of RGS19 in HEK293 cells appeared to stimulate Akt signaling but impair the Ras/Raf/MEK/ERK cascade [19]. The opposing actions on Akt and Ras pathways might account for the ability of RGS19 to stimulate cell proliferation without inducing neoplastic transformation [17]. Here, we examined the effect of RGS19 expression on Ras signaling. Our results suggest that RGS19 inhibits Ras signaling through a complex network involving a scaffold protein KSR1 and a nucleoside diphosphate kinase Nm23.