Rit, a non-lipid-modi®ed Ras-related protein, transforms NIH3T3 cells without activating the ERK, JNK, p38 MAPK or PI3K/Akt pathways Elena V Rusyn 1 , Evangeline R Reynolds 1 , Haipeng Shao 3 , Theresa M Grana 1,2 , Tung O Chan 4 , Douglas A Andres 3 and Adrienne D Cox* ,1,2 1 Departments of Radiation Oncology and Pharmacology, CB 7512, Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, NC 27599, USA; 2 Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, NC 27599, USA; 3 Department of Biochemistry, University of Kentucky, Lexington, Kentucky, KY 40536, USA; 4 Kimmel Cancer Center, Thomas Jeerson University, Philadelphia, PA 19107, USA 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 *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 con- structs regulated by SRF, NF-kB, 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 farnesyltransfer- ase 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 eector pathways in NIH3T3 ®broblasts for this neural-speci®c Ras family member. In summary, although Rit shares most core eector domain residues with Ras, our results suggest that Rit uses novel eector pathways to regulate proliferation and transformation. Oncogene (2000) 19, 4685 ± 4694. Keywords: Rit; Ras; signaling; transformation; lipid modi®cation; farnesyltransferase Introduction Ras-related small GTPases act as guanine nucleotide binding switches that regulate a wide variety of cell functions including cell proliferation, dierentiation and apoptosis (Campbell et al., 1998; Reuther and Der, 2000). Within the Ras subfamily are branches with transforming capability, including those of Ras and R-Ras, and those that are nontransforming, including those of Rap, Ral, and Rheb. Members of these branches of the Ras subfamily are covalently modi®ed by farnesyl or geranylgeranyl isoprenoid lipids that are the ®rst step in targeting to cellular membranes, and both their lipid modi®cation and their correct subcellular localization are required for proper biological function (Cox and Der, 1992). In addition, such proteins also require modi®cation by palmitic acid acylation for full activity (Hancock et al., 1990). Recently, a novel branch of the Ras subfamily has been described, whose members share highly conserved GTP binding motifs but do not contain any known lipidation motifs. Members of this novel branch include Rit (Ras-like protein in tissues), Rin (Ras-like protein in neurons) and the Drosophila protein RIC (Ras-related protein which interacted with calmodulin). Dendrograms of the Ras family show that Rit and Rin form a distinct subfamily more highly related to the Ras and R-Ras branches than to the Rap, Ral or Rheb branches (Campbell et al., 1998). The conservation of Rit and Rin from ¯ies to humans suggests conservation of important physiological functions. Rit proteins have been cloned independently by groups looking for calmodulin binding proteins (Wes et al., 1996), retinally expressed Ras-related proteins (Lee et al., 1996) and, fortuitously, retinally expressed farnesylated proteins (Shao et al., 1999). Unique features of this branch include calmodulin binding for the neuronally expressed forms, lack of prenyla- tion or other lipidation signals in all members, and a distinct but conserved G2 core eector domain (HDPTIEDAY) in which histidine is substituted for tyrosine at position 32, and alanine is substituted for serine at position 39. This level of similarity to other Ras family members suggests that Rit proteins likely possess distinct but partially overlapping functions with other branches of the Ras family. No character- ization of their cellular functions has yet been performed. As an initial step in the biological characterization of Rit and Rin, we investigated their ability to regulate cell growth, transformation, and several signaling pathways used by other Ras family proteins. We found that Rit signaled to Ras- responsive elements and transformed NIH3T3 cells to tumorigenicity, but failed to activate the ERK, JNK, p38 or PI3-K/Akt kinases, indicating that Rit regulates growth control by dierent eector path- ways than the other transforming members of the Ras family. Oncogene (2000) 19, 4685 ± 4694 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc *Correspondence: D Cox Received 17 March 2000; revised 17 June 2000; accepted 1 August 2000