Elsevier

Cellular Signalling

Volume 22, Issue 3, March 2010, Pages 366-376
Cellular Signalling

RGS14 is a multifunctional scaffold that integrates G protein and Ras/Raf MAPkinase signalling pathways

https://doi.org/10.1016/j.cellsig.2009.10.005Get rights and content

Abstract

MAPkinase signalling is essential for cell growth, differentiation and cell physiology. G proteins and tyrosine kinase receptors each modulate MAPkinase signalling through distinct pathways. We report here that RGS14 is an integrator of G protein and MAPKinase signalling pathways. RGS14 contains a GPR/GoLoco (GL) domain that forms a stable complex with inactive Giα1/3–GDP, and a tandem (R1, R2) Ras binding domain (RBD). We find that RGS14 binds and regulates the subcellular localization and activities of H-Ras and Raf kinases in cells. Activated H-Ras binds RGS14 at the R1 RBD to form a stable complex at cell membranes. RGS14 also co-localizes with and forms a complex with Raf kinases in cells. The regulatory region of Raf-1 binds the RBD region of RGS14, and H-Ras and Raf each facilitate one another's binding to RGS14. RGS14 selectively inhibits PDGF-, but not EGF- or serum-stimulated Erk phosphorylation. This inhibition is dependent on H-Ras binding to RGS14 and is reversed by co-expression of Giα1, which binds and recruits RGS14 to the plasma membrane. Giα1 binding to RGS14 inhibits Raf binding, indicating that Giα1 and Raf binding to RGS14 are mutually exclusive. Taken together, these findings indicate that RGS14 is a newly appreciated integrator of G protein and Ras/Raf signalling pathways.

Introduction

Heterotrimeric G proteins (G proteins) are molecular switches that regulate a broad range of signalling events important for many aspects of cell and organ physiology. The regulators of G protein signalling (RGS proteins) inactivate Gα-GTP subunits to negatively regulate G protein signalling. In conventional signalling models [1], G proteins link cell surface receptors of neurotransmitters and hormones to intracellular signalling events, and RGS proteins limit the lifetime of those signalling events. RGS proteins are defined by a shared 130 amino acid RGS domain which binds Gα-GTP to act as a GTPase activating protein (GAP) and inhibit G protein signalling [2], [3], [4]. Family members are classified into six or more distinct subgroups based on amino acid sequence identities and functional similarities. Many are relatively small (20–30 kDa) simple proteins with unremarkable features outside of the RGS domain, while others are larger (60–160 kDa) more “complex” proteins with additional domains that bind other protein partners to confer distinct signalling functions and/or act as novel integrators of G protein signalling.

RGS14 is a highly unusual complex family member that contains a canonical RGS domain, a tandem (R1 and R2) Ras/Rap binding domain (RBD) [5], [6], and a GoLoco/GPR (GL) motif. RGS14 was originally identified as a novel binding partner for the Ras-like GTPases, Rap1/2 [7]. The full-length sequence [8] encodes a ~ 62 kDa protein most closely related to RGS12 (85 kDa) and RGS10 (22 kDa). We and others show that RGS14 interacts selectively with the Gi/o subfamily of G proteins to regulate their guanine nucleotide binding/hydrolysis activity and signalling functions [9], [10], [11], [12], [13]. The RGS domain binds directly to activated Gi/oα-GTP to confer non-selective GAP activity towards Giα and Goα. By contrast, we find that the GoLoco/GPR (GL) motif binds specifically to inactive Giα1–GDP and Giα3–GDP, but not Giα2 or Goα, to act as an inhibitor of GDP dissociation (GDI) much like Gβγ subunits [10], [12], [13]. Furthermore, we have shown that RGS14 is actively recruited from the cytosol to the plasma membrane by inactive Giα1–GDP or Giα3–GDP [14]. This recruitment is mediated by the GL domain of RGS14, and Giα1/RGS14 form a stable complex at the plasma membrane in cells. Other known Gα/GL protein complexes are thought to be involved with unconventional G protein signalling pathways [15], [16], [17] that rely on the combined action of non-receptor guanine nucleotide exchange factors (GEFs) and GL proteins in place of Gβγ subunits to regulate and mediate Gα signalling. These pathways have been shown to be important for cell division, neuronal development and synaptic plasticity in both lower and higher eukaryotes [16], [18]. Thus, RGS14 may serve new functions that can be attributed to the action of its poorly understood GL domain distinct from those contributed by its RGS GAP domain.

Both G proteins and tyrosine kinase growth factor receptors activate MAPkinase signalling by distinct pathways [19], [20], [21]. MAPkinase signalling is important for many aspects of cell physiology including cell growth, survival, and differentiation as well as neuronal development and synaptic plasticity [22]. Since RGS14 tightly binds inactive forms of Giα1/3 and also contains tandem (R1 and R2) Ras/Rap binding domains (RBD), we investigated whether RGS14 could integrate the actions of Giα, Ras and Raf kinases to regulate MAP kinase signalling. We report that RGS14 binds activated H-Ras and its effectors Raf-1, B-Raf and A-Raf to regulate PDGF-stimulated Erk phosphorylation, and that this activity of RGS14 is regulated by its interactions with Giα1. RGS14 is recruited by H-Ras to form a stable complex at the plasma membrane, and it binds both H-Ras and/or Raf kinases either alone or together in cells. Expression of RGS14 inhibits PDGF-stimulated Erk phosphorylation and this inhibition is dependent on H-Ras binding, but is reversed by Giα1 which also blocks Raf binding to RGS14. Our findings demonstrate that RGS14 serves as a novel scaffold to integrate Giα1 and Ras/Raf/MAPkinase signalling events through the action of its GL domain.

Section snippets

Plasmids and antibodies

The RGS14 cDNA used in this study was derived from rat (Genbank accession number of U92279) as previously described [10]. Truncated Flag-Raf-1 (ΔRaf-1) and 3xHA-B-Raf were kindly supplied by Dr. Haian Fu (Emory University). 3xHA-A-Raf cDNA was kindly supplied by Dr. Deborah Anderson (Saskatchewan Cancer Agency). HA-Raf-1 was kindly supplied by Dr. Ulf R. Rapp (Würzburg University). Glu–Glu epitope (EE) tagged recombinant Giα1/pcDNA3.1 plasmids, H-Ras 3xHA-tagged (N-terminus) plasmids and their

RGS14 binds to and co-localizes with activated H-Ras in cells

RGS14 was first identified as a binding partner for the Ras-like GTPase, Rap1/2 [7]. Ras and Rap each specifically bind RBD domains and RGS14 contains tandem (R1 and R2) RBDs [7], [25]. We tested if RGS14 could bind H-Ras. RGS14 (Flag-RGS14) was transfected together with different forms of Ras (HA-Ras) in HeLa cells. We observed that RGS14 bound to and co-immunoprecipitated with constitutively active (G/V mutant) H-Ras, but only weakly to wild type H-Ras and not at all to inactive (S/N mutant)

Discussion

RGS14 is a highly unusual signalling protein that contains two distinct Gα interaction sites (RGS domain and GL domain) and tandem (R1 and R2) RBD domains that are known in other proteins to bind Ras and Rap GTPases. Indeed, RGS14 was first identified as a novel Rap1/2 binding partner [7]. We and others have shown previously that the RGS domain of RGS14 is a promiscuous and non-selective GAP for active forms of Giα and Goα, and that the GL domain is a selective GDI for Giα, but not Goα [7], [10]

Conclusion

We report that RGS14 binds activated H-Ras and Raf-1 kinases, both independently and together in cells. RGS14 inhibits PDGF stimulated MAPkinase activity in a H-Ras dependent manner. This inhibition of signalling and Raf-1 binding to RGS14 is reversed by Giα1, suggesting that Giα1 binding and Raf-1 binding to RGS14 are mutually exclusive. Giα1 expression in cells also recruits Raf-1 to the plasma membrane suggesting a larger Gi-regulated signalling complex. Together, our findings here suggest

Acknowledgements

We thank Dr. Ulf R. Rapp (Wϋrzburg University), Dr. Haian Fu (Emory University) and Dr. Deborah Anderson (Saskatchewan Cancer Agency) for kindly sharing cDNA constructs used in these studies, as described in the Experimental Procedures section. We also thank Jon Waters for technical assistance in generating some mutant constructs. This work is supported by grants from the National Institutes of Health awarded to J.R.H. (R01NS37112 and R01NS049195).

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