Analysis of the Biochemical and Biological Activities of Deletion Mutants of the H-Ras P21 Protein Suggest That Gap is an Essential Component of Its Effector Function
The anti-ras p21 monoclonal antibody Y13–259 has been shown to efficiently neutralize DNA synthesis induced by serum in quiescent cells (1), and phenotypically reverts cells transformed by a variety of oncogenes (2). Due to the putative biological relevance of the epitope recognized by the monoclonal antibody Y13–259, we have generated deletion mutants of the H-ras p21 protein which lack residues 58 to 63 or 64 to 68, and contain either glycine or arginine at position 12. In addition, those mutants carrying a deletion at position 64 to 68 also carried an activating substitution of Thr at position 59. None of the deleted proteins were recognized by monoclonal antibody Y13–259, and those mutants carrying activating mutations showed at least a 100-fold reduction in their transforming activities compared to their non-deleted counterparts. The mutant proteins carrying a normal 12 position also showed a decreased transforming activity when compared to the normal protein. Alterations observed in the in vitro GTPase or GTP-interchange properties in our deletion mutants were not consistent with their decreased transforming activities. Moreover, all the generated mutants showed normal palmitylation and membrane localization, known to be essential for biological activity of ras proteins. Recently, a protein, designated as GAP, has been described which is able to specifically increase the GTPase activity of normal ras. p21 (3). We have found that GAP is unable to increase the GTPase activity of our deleted proteins. These observations strongly suggest that the recognition site for Y13–259 within the ras p21 molecule influences directly or indirectly the interaction of ras p21 with GAP, and suggest that this interaction is critical for the biological activity of ras. proteins.
KeywordsDeletion Mutant GTPase Activity Transforming Activity Delete Protein Abelson Murine Leukemia Virus
Unable to display preview. Download preview PDF.
- 5..Lacal, J.C., and Tronick, S.E. The ras oncogene. In The Oncogene Handbook Reddy, P, Curran, T., and Skalka, A. edts. Elsvier, Holland (in press).Google Scholar
- 33.Colby, W.W., Hayflick, J.S., Clark, S.G., and Levinson, A.D. Mol. Cell. Bio1.6, 730–734 (1986).Google Scholar