A RAS EFFECTOR DOMAIN MUTANT WHICH IS TEMPERATURE SENSITIVE FOR CELLULAR-TRANSFORMATION - INTERACTIONS WITH GTPASE-ACTIVATING PROTEIN AND NF-1

被引:34
作者
DECLUE, JE
STONE, JC
BLANCHARD, RA
PAPAGEORGE, AG
MARTIN, P
ZHANG, K
LOWY, DR
机构
[1] NCI,CELLULAR ONCOL LAB,BLDG 37,ROOM 1B-26,BETHESDA,MD 20892
[2] UNIV ALBERTA,DEPT BIOCHEM,EDMONTON T6G 2H7,ALBERTA,CANADA
关键词
D O I
10.1128/MCB.11.6.3132
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A series of v-ras(H) effector domain mutants were analyzed for their ability to transform rat 2 cells at either low or high temperatures. Three mutants were found to be significantly temperature sensitive: Ile-36 changed to Leu, Ser-39 changed to Cys (S39C), and Arg-41 changed to Leu. Of these, the codon 39 mutant (S39C) showed the greatest degree of temperature sensitivity. When the same mutation was analyzed in the proto-oncogene form of ras (c-ras(H)), this gene was also found to be temperature sensitive for transformation. Biochemical analysis of the proteins encoded by v-ras(H)(S39C) and c-ras(H)(S39C) demonstrated that the encoded p21ras proteins were stable and bound guanine nucleotides in vivo at permissive and nonpermissive temperatures. On the basis of these findings, it is likely that the temperature-sensitive phenotype results from an inability of the mutant (S39C) p21ras to interact properly with the ras target effector molecule(s) at the nonpermissive temperature. We therefore analyzed the interaction between the c-ras(H)(S39C) protein and the potential target molecules GTPase-activating protein (GAP) and the GAP-related domain of NF-1, on the basis of stimulation of the mutant p21ras GTPase activity by these molecules in vitro. Assays conducted across a range of temperatures revealed no temperature sensitivity for stimulation of the mutant protein, compared with that of authentic c-ras(H) protein. We conclude that for this mutant, there is a dissociation between the stimulation of p21ras GTPase activity by GAP and the GAP-related domain NF-1 and their potential target function. Our results are also consistent with the existence of a distinct, as-yet-unidentified effector for mammalian ras proteins.
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页码:3132 / 3138
页数:7
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