Rap1 Activation in Response to cAMP Occurs Downstream of Ras Activation during Dictyostelium Aggregation

Parvin Bolourani,George B Spiegelman,Gerald Weeks

doi:10.1074/jbc.m707459200

Parvin Bolourani, George B Spiegelman + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m707459200

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Abstract

We have used a doubly disrupted rasC(-)/rasG(-) strain of Dictyostelium discoideum, which ectopically expresses the carA gene, to explore the relationship between the activation of RasC and RasG, the two proteins that are necessary for optimum cAMP signaling, and the activation of Rap1, a Ras subfamily protein, that is also activated by cAMP. The ectopic expression of carA restored early developmental gene expression to the rasC(-)/rasG(-) strain, rendering it suitable for an analysis of cAMP signal transduction. Because there was negligible signaling through both the cAMP chemotactic pathway and the adenylyl cyclase activation pathway in the rasC(-)/rasG(-)/[act15]:carA strain, it is clear that RasG and RasC are the only two Ras subfamily proteins that directly control these pathways. The position of Rap1 in the signal transduction cascade was clarified by the finding that Rap1 activation was totally abolished in rasC(-)/rasG(-)/[act15]:carA and rasG(-) cells but only slightly reduced in rasC(-) cells. Rap1 activation, therefore, occurs downstream of the Ras proteins and predominantly, if not exclusively, downstream of RasG. The finding that in vitro guanylyl cyclase activation is also abolished in the rasC(-)/rasG(-)/[act15]:carA strain identifies RasG/RasC as the presumptive monomeric GTPases required for this activation.

Highlights

10232 JOURNAL OF BIOLOGICAL CHEMISTRY discoideum [3, 4] has raised important questions regarding the specific functions of individual Ras proteins
The aggregation process is driven in response to cAMP, which is synthesized and secreted soon after the onset of starvation. cAMP binds to cell surface cAMP receptors, resulting in the dissociation and activation of a heterotrimeric G protein
This leads to chemotaxis to cAMP and to the activation of adenylyl cyclase A (ACA) [8]

Summary

EXPERIMENTAL PROCEDURES

Dictyostelium Strains—Generation of rasGϪ, rasCϪ, and rasCϪ/rasGϪ strains, all in the JH10 background, has been described previously [10]. CAMP-pulsed cells were washed twice in KK2, resuspended to 5 ϫ 107 cells/ml in KK2, and stimulated by addition of cAMP to a final concentration of 100 nM. CGMP Production and Guanylyl Cyclase Activation—To measure cGMP production, cells were pulsed with cAMP as described above, washed twice with KK2, and resuspended to a density of 1 ϫ 108 cells/ml in KK2 containing 2 mM caffeine. The lysates were centrifuged for 10 min, and the protein concentrations of the supernatants were determined using the protein assay from Bio-Rad. 400 ␮g of protein was incubated with 100 ␮g of GST-Byr2-RBD on glutathione-Sepharose beads (GE Healthcare Bio-sciences AB, Uppsala, Sweden) at 4 °C for 1 h. Samples were subjected to SDS-PAGE and Western blot analysis with anti-RasG, RasC, or Rap antibodies

RESULTS

Number of Instantaneous Chemotaxis cells velocity indexa

DISCUSSION