Ras isoforms: signaling specificities in CD40 pathway

Arathi Nair,Late Anirban Banerji,Bhaskar Saha,Ankita Srivastava,Charudutta Navare,Sushmita Chakraborty

doi:10.1186/s12964-019-0497-1

Abstract

BackgroundRas are small cellular GTPases which regulate diverse cellular processes. It has three isoforms: H-Ras, K-Ras, and N-Ras. Owing to the N-terminus (1–165 residues) sequence homology these isoforms were thought to be functionally redundant. However, only K-Ras-deficient mice but not H-Ras- and N-Ras-deficient mice show embryonic lethality. Similarly, mutations in a given Ras isoform are associated with a particular type of cancer. Moreover, we have previously reported that Ras isoforms perform unique functions in Leishmania major infection. Thus, Ras isoforms are implicated to have signaling and functional specificity but the mechanism remains to be elucidated.ResultUsing CD40 as a model receptor, we showed that depending on the strength of signaling, specific Ras isoforms are activated. Weak CD40 signal activates N-Ras, whereas strong signal activates H-Ras and K-Ras. Additionally, we showed that suppression of N-Ras expression reduced CD40-induced extracellular signal–regulated kinase-1/2 (ERK-1/2) activation and Interleukin (IL)-10 production; whereas suppression of H-Ras or K-Ras reduced CD40-induced p38 mitogen-activated protein kinase (p38MAPK) activation and IL-12 production. Furthermore, we showed that Ras isoforms have activator (GEF) specificity as weak CD40 signal-activated N-Ras requires Sos-1/2 whereas strong CD40 signal-activated H-Ras/K-Ras requires Ras-GRP as the guanine-nucleotide exchange factor (GEF) inducing ERK-1/2- or p38MAPK-mediated IL-10 or IL-12 productions, respectively, in macrophages. Silencing of syk reduced CD40-induced N-Ras activation but silencing of lyn inhibited H-Ras and K-Ras activation. In CD40 signaling, Ras isoforms also showed effector specificity; while H-Ras and K-Ras showed specificity for phosphatidyl inositol-3 kinase activation at high dose of CD40 stimulation, N-Ras primarily associated with Raf-1 at low dose of CD40 stimulation. Moreover, fractal analysis showed that functional site surface roughness for H-Ras (SurfaceFD = 2.39) and K-Ras (SurfaceFD = 2.39) are similar but significantly different from N-Ras (SurfaceFD = 2.25).ConclusionThe activator and effector specificities of Ras isoforms in CD40 signaling indicated their differential involvement in CD40 pathway and in maintaining the reciprocity. Our observations reveal Ras-regulated signaling outcome and its potential for developing Ras isoform-targeted immunotherapy and prophylaxis.Graphical abstract

Highlights

Ras are small cellular GTPases which regulate diverse cellular processes
Ras isoforms are differentially activated in CD40 signaling Previously we have shown that macrophage expressed CD40, induces activation of extracellular signal–regulated kinase-1/2 (ERK-1/2)-mediated anti-inflammatory IL-10 production and p38 mitogen-activated protein kinase (p38MAPK)-mediated pro-inflammatory IL-12 production, depending on the strength of signaling [11] reflecting a functional duality for CD40
In order to decipher signaling specificity of Ras isoforms in a receptor-driven system, we utilized reciprocal CD40 signaling in macrophages as a model and examined the activation of Ras isoforms

Summary

Introduction

Ras are small cellular GTPases which regulate diverse cellular processes It has three isoforms: H-Ras, KRas, and N-Ras. Owing to the N-terminus (1–165 residues) sequence homology these isoforms were thought to be functionally redundant. K-Ras mutation is more frequently observed in pancreatic and colon cancers while N-Ras mutation is observed in acute leukemia [3] These observations associate mutated Ras isoforms with particular tissues only during metastatic transformation, one might imply these isoforms in non-transformed tissuespecific functions. Germ-line mutational studies suggest that H-Ras mutation is observed in more than 86% in Costello syndrome cases [7], whereas K-Ras mutation is frequently observed in cardio-facial-cutaneous (CFC) syndrome [8], suggesting isoform specific association with these syndromes These observations describe differential involvements of Ras isoforms in oncogenesis or embryonic development and imply functional non-redundancy in Ras isoforms. As the catalytic domain in Ras protein lies in the conserved Gdomain, the differences in their functions and any signaling specificity remains a paradox

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