Cyclic AMP signalling in Dictyostelium: G-proteins activate separate Ras pathways using specific RasGEFs

Abstract

In general, mammalian Ras guanine nucleotide exchange factors (RasGEFs) show little substrate specificity, although they are often thought to regulate specific pathways. Here, we provide in vitro and in vivo evidence that two RasGEFs can each act on specific Ras proteins. During Dictyostelium development, RasC and RasG are activated in response to cyclic AMP, with each regulating different downstream functions: RasG regulates chemotaxis and RasC is responsible for adenylyl cyclase activation. RasC activation was abolished in a gefA- mutant, whereas RasG activation was normal in this strain, indicating that RasGEFA activates RasC but not RasG. Conversely, RasC activation was normal in a gefR- mutant, whereas RasG activation was greatly reduced, indicating that RasGEFR activates RasG. These results were confirmed by the finding that RasGEFA and RasGEFR specifically released GDP from RasC and RasG, respectively, in vitro. This RasGEF target specificity provides a mechanism for one upstream signal to regulate two downstream processes using independent pathways.

Figure 1

gefA⁻ mutants are deficient in RasC activation. Six-hour-pulsed A×3 and gefA⁻ cells were stimulated by the addition of 200 nM cyclic AMP, samples were collected at the indicated time points after stimulation and the lysates from the cells were subjected to the RBD-binding assay. Samples were fractionated by SDS–PAGE, transferred onto polyvinylidine fluoride (PVDF) membrane and probed with RasC-specific, RasG-specific or a pan-specific Ras antibody, as indicated. This figure is representative of three independent experiments. gef, guanine-nucleotide exchange factor; RBD, Ras-binding domain; SDS–PAGE, SDS–polyacrylamide gel electrophoresis.

Figure 2

Loss of RasGEFR reduces activation of RasG during development. Six-hour-pulsed (A) gefR⁻, (B) gefC⁻ and (C) gefD⁻ cells were stimulated by the addition of 200 nM cyclic AMP and assayed for activated Ras as described in Fig 1. This figure is representative of two independent experiments. gef, guanine-nucleotide exchange factor.

Figure 3

RasGEFA stimulates GDP release from RasC but not RasD or RasG. Ras proteins were loaded with mouse GDP, incubated with (filled squares) or without (filled circles) 1 μM GefA^CDC25, or with (filled triangles) 1 μM CDC25, and GDP release was plotted as a decrease in relative fluorescence with time. The curves shown are (A) RasC^CT, (B) RasG and (C) RasD. gef, guanine-nucleotide exchange factor.

Figure 4

RasGEFR stimulates the release of GDP from RasG but not RasC or RasD. Ras proteins were loaded with mouse GDP, incubated with (filled squares) or without (filled circles) 1 μM GefR^CDC25, or with (filled triangles) 1 μM CDC25, and GDP release was plotted as a decrease in relative fluorescence with time. The curves shown are (A) RasC^CT, (B) RasG and (C) RasD. gef, guanine-nucleotide exchange factor.