Rit, a non-lipid-modified Ras-related protein, transforms NIH3T3 cells without activating the ERK, JNK, p38 MAPK or PI3K/Akt pathways.
The biological functions of Rit (Ras-like protein in tissues) and Rin (Ras-like protein in neurons), members of a novel branch of Ras-related GTP-binding proteins that are approximately 50% identical to Ras, have not been characterized. Therefore, we assessed their activity in growth control, transformation and signaling. NIH cells stably expressing a constitutively activated mutant of Rit [Rit(79L)] (analogous to the oncogenic mutant H-Ras(61L)) demonstrated strong growth transformation, proliferating rapidly in low serum and forming colonies in soft agar and tumors in nude mice. Although Rit(79L) alone did not promote morphologically transformed foci, it cooperated with both Raf and Rho A to form Rac/Rho-like foci. Rin [Rin(78L)] cooperated only with Raf. Rit(79L) but not Rin(78L) stimulated transcription from luciferase reporter constructs regulated by SRF, NF-kappaB, Elk-1 and Jun. However, neither activated ERK, JNK or p38, or PI3-K/Akt kinases in immune complex kinase assays. Interestingly, although Rit lacks any known recognition signal for C-terminal lipidation, Rit-transformed cell growth and survival in low serum is dependent on a farnesylated protein, as treatment with farnesyltransferase inhibitors caused apoptosis. Rin cooperated with Raf in focus assays but did not otherwise function in these assays, perhaps due to a lack of appropriate effector pathways in NIH3T3 fibroblasts for this neural-specific Ras family member. In summary, although Rit shares most core effector domain residues with Ras, our results suggest that Rit uses novel effector pathways to regulate proliferation and transformation.
Rusyn
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Department of Radiation Oncology, Lineberger Cancer Center, University of North Carolina at Chapel Hill, 27599, USA.
Reynolds
E R
ER
Shao
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H
Grana
T M
TM
Chan
T O
TO
Andres
D A
DA
Cox
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AD
D016475:3T3 Cells / Q000637:transplantation; D000818:Animals; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D003260:Contact Inhibition; D016895:Culture Media, Serum-Free; D004268:DNA-Binding Proteins / Q000235:genetics; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology*; D016758:Genes, jun; D048031:JNK Mitogen-Activated Protein Kinases; D020935:MAP Kinase Signaling System; D008715:Methionine / Q000031:analogs & derivatives / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D019950:Mitogen-Activated Protein Kinase 1 / Q000502:physiology; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D016328:NF-kappa B / Q000235:genetics; D009368:Neoplasm Transplantation; D009687:Nuclear Proteins / Q000235:genetics; D010641:Phenotype; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D017368:Protein Prenylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf / Q000502:physiology; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics; D036224:Receptor, EphB4; D036081:Receptors, Eph Family; D026362:Serum Response Factor; D015398:Signal Transduction; D014410:Tumor Stem Cell Assay; D048051:p38 Mitogen-Activated Protein Kinases; D018631:ras Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D020742:rhoA GTP-Binding Protein / Q000502:physiology
D016895:Culture Media, Serum-Free; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D048031:JNK Mitogen-Activated Protein Kinases; D008715:Methionine; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D016328:NF-kappa B; D009687:Nuclear Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf; D020794:Receptor Protein-Tyrosine Kinases; D036224:Receptor, EphB4; D036081:Receptors, Eph Family; D026362:Serum Response Factor; D048051:p38 Mitogen-Activated Protein Kinases; D018631:ras Proteins; D020742:rhoA GTP-Binding Protein; C096856:FTI 277; C096898:L 744832; C401848:Rin protein (GTPase); C472865:Rit1 protein, mouse