Biochemical characterization of the Ras-related GTPases Rit and Rin.
We report the biochemical characterization of Rit and Rin, two members of the Ras superfamily identified by expression cloning. Recombinant Rit and Rin bind GTP and exhibit intrinsic GTPase activity. Conversion of Gln to Leu at position 79 (for Rit) or 78 (for Rin) (equivalent to position 61 in Ras) resulted in a complete loss of GTPase activity. Surprisingly, significant differences were found when the guanine nucleotide dissociation constants of Rit and Rin were compared with the majority of Ras-related GTPases. Both proteins display higher k(off) values for GTP than GDP in the presence of 10 mM Mg(2+). These GTP dissociation rates are 5- to 10-fold faster than most Ras-like GTPases. Despite these unique biochemical properties, our data support the notion that both Rit and Rin function as nucleotide-dependent molecular switches. To begin to address whether these proteins act as regulators of distinct signaling pathways, we examined their interaction with a series of known Ras-binding proteins by yeast two-hybrid analysis. Although Rit, Rin, and Ras have highly related effector domain sequences, Rit and Rin were found to interact with the known Ras binding proteins RalGDS, Rlf, and AF-6/Canoe but not with the Raf kinases, RIN1, or the p110 subunit of phosphatidylinositol 3-kinase. These interactions were GTP and effector domain dependent and suggest that RalGDS, Rlf, and AF-6 are Rit and Rin effectors. Their biochemical properties and interaction with a subset of known Ras effector proteins suggest that Rit and Rin may play important roles in the regulation of signaling pathways and cellular processes distinct from those controlled by Ras.
Shao
H
H
Department of Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536-0084, USA.
Kadono-Okuda
K
K
Finlin
B S
BS
Andres
D A
DA
D003001:Cloning, Molecular; D020662:Guanine Nucleotide Exchange Factors; D006150:Guanine Nucleotides / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D016547:Kinesins / Q000378:metabolism; D008969:Molecular Sequence Data; D009218:Myosins / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D020726:ral Guanine Nucleotide Exchange Factor / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
C095516:AFDN protein, human; C490657:Afdn protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D006150:Guanine Nucleotides; C097542:RLF protein, human; D011994:Recombinant Proteins; C490805:Rgl2 protein, mouse; D014157:Transcription Factors; D020726:ral Guanine Nucleotide Exchange Factor; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; C486776:RIT1 protein, human; C401848:Rin protein (GTPase); D009218:Myosins; D016547:Kinesins; D018631:ras Proteins