Figure 2.

Interaction of Dictyostelium RIP3 with different Ras proteins. (A) Amino acid sequence comparison of Dictyostelium RasG, RasD, and RasB and human Ha-Ras. The asterisks indicate positions of conservation between RasG and Ha-Ras but not RasD. (B) The carboxyl-terminal region of RIP3 that was identified and cloned in two-hybrid screens using mammalian Ha-Ras^G12V as bait was used in two-hybrid assays to examine interaction of this domain with the activated form of the five identified Dictyostelium Ras proteins (RasG, RasD, RasB, RasC, and RasS). In addition, the dominant negative or nonactivatable form of RasG (RasG^S17N) and interaction with Ha-Ras^G12V and Ha-Ras^G15A is shown. As controls, the interactions of the previously identified Dictyostelium IQGAP and the related gene RasGAPa as well as the Ras-interacting domain of Dictyostelium P110-related PI3 kinase DdPI3K1 are shown. The level of two-hybrid interactions was quantitated by the level of β-galactosidase production and the intensity of blue staining of yeast colonies.