RIG1 inhibits the Ras/mitogen-activated protein kinase pathway by suppressing the activation of Ras.
The retinoid-inducible gene 1 (RIG1) protein is a retinoid-inducible growth regulator. Previous studies have shown that the RIG1 protein inhibits the signaling pathways of Ras/mitogen-activated protein kinases. However, neither the mode of action nor the site of inhibition of RIG1 is known. This study investigated the effects of RIG1, and the mechanisms responsible for these effects, on the activation of Ras proteins in HtTA cervical cancer cells. RIG1 reduced the levels of activated Ras (Ras-GTP) and total Ras protein in cells transfected with mutated H-, N-, or K-Ras(G12V), or in cells transfected with the wild type H- or N-Ras followed by stimulation with epidermal growth factor. The half-life of Ras protein decreased from more than 36 h in control cells to 18 h in RIG1-transfected cells. RIG1 immunoprecipitated with the Ras protein in co-transfected cellular lysates. In contrast to the predominant plasma membrane localization in control cells, the H-Ras fusion protein EGFP-H-Ras was localized within a discrete cytoplasmic compartment where it co-localized with RIG1. RIG1 inhibited more than 93% of the Elk- and CHOP-mediated transactivation induced by H- or K-Ras(G12V). However, RIG1 did not inhibit the transactivation induced by MEK1 or MEK3, and failed to suppress the phosphorylation of extracellular signal-regulated kinases 1 and 2 induced by the constitutively activated B-Raf(V599E). The RIG1 with carboxyl terminal truncation (RIG1DeltaC) did not immunoprecipitate with Ras and had no effect on Ras activation or transactivation of the downstream signal pathways. These data indicate that RIG1 exerts its inhibitory effect at the level of Ras activation, which is independent of Ras subtype but dependent on the membrane localization of the RIG1 protein. This inhibition of Ras activation may be mediated through downregulation of Ras levels and alteration of Ras subcellular distribution.
Tsai
Fu-Ming
FM
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan.
Shyu
Rong-Yaun
RY
Jiang
Shun-Yuan
SY
D015153:Blotting, Western; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases; D015536:Down-Regulation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005260:Female; D011905:Genes, ras; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D048369:MAP Kinase Kinase 1 / Q000502:physiology; D048371:MAP Kinase Kinase 3 / Q000502:physiology; D020935:MAP Kinase Signaling System / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009154:Mutation; D054512:Phospholipases A2, Calcium-Independent; D010766:Phosphorylation; D020033:Protein Isoforms; D011506:Proteins / Q000378:metabolism; D020365:RNA Helicases / Q000737:chemistry / Q000378:metabolism*; D011971:Receptors, Immunologic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D014162:Transfection; D025521:Tumor Suppressor Proteins; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D047908:Intracellular Signaling Peptides and Proteins; D020033:Protein Isoforms; D011506:Proteins; D011971:Receptors, Immunologic; D025521:Tumor Suppressor Proteins; D004815:Epidermal Growth Factor; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D048371:MAP Kinase Kinase 3; C482125:MAP2K1 protein, human; C482530:MAP2K3 protein, human; C490065:PLAAT3 protein, human; D054512:Phospholipases A2, Calcium-Independent; C452820:RIGI protein, human; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases; D018631:ras Proteins