Protein kinase A regulates the Ras, Rap1 and TORC2 pathways in response to the chemoattractant cAMP in Dictyostelium

Abstract

Efficient directed migration requires tight regulation of chemoattractant signal transduction pathways in both space and time, but the mechanisms involved in such regulation are not well understood. Here, we investigated the role of protein kinase A (PKA) in controlling signaling of the chemoattractant cAMP in Dictyostelium discoideum. We found that cells lacking PKA display severe chemotaxis defects, including impaired directional sensing. Although PKA is an important regulator of developmental gene expression, including the cAMP receptor cAR1, our studies using exogenously expressed cAR1 in cells lacking PKA, cells lacking adenylyl cyclase A (ACA) and cells treated with the PKA-selective pharmacological inhibitor H89, suggest that PKA controls chemoattractant signal transduction, in part, through the regulation of RasG, Rap1 and TORC2. As these pathways control the ACAmediated production of intracellular cAMP, they lie upstream of PKA in this chemoattractant signaling network. Consequently, we propose that the PKA-mediated regulation of the upstream RasG, Rap1 and TORC2 signaling pathways is part of a negative feedback mechanism controlling chemoattractant signal transduction during Dictyostelium chemotaxis.

Caffeine inhibits PI3K and mTORC2 in Dictyostelium and differentially affects multiple other cAMP chemoattractant signaling effectors

“…Phospho-ERK1 and -ERK2 were detected using phospho-p44/42 MAPK antibody diluted 1:5000. PKB and PKA substrates phosphorylation was detected using phospho-Akt substrate and phospho-(Ser/Thr) PKA antibodies, respectively, diluted 1:2000 as described previously [17].…”

“…and that Protein Kinase A (PKA), which is activated by intracellular cAMP, negatively regulates the activity of upstream RasG, Rap1, and mTORC2 pathways ( Fig. 1) [16,17].…”

“…antibodies, Lifeact-GFP, GFP-MyoII, and PHcrac-GFP constructs were gifts from Rick Firtel and were previously described [19][20][21][22]. Flag-tagged RasC construct was reported elsewhere [17]. T7-tagged Pianissimo (T7-Pia) was cloned by ligating AvrII digested restriction sites into the compatible SpeI site of the extrachromosomal vector pDM304, which was obtained from the Dicty Stock Center [23] (depositor: Douwe Veltman;…”

The physiological regulation of macropinocytosis duringDictyosteliumgrowth and development

“…This can be a confounding influence on studies of 491 chemotaxis, since macropinocytosis uses the same actin machinery as 492 pseudopods and thus impairs chemotaxis(Veltman, 2015). In particular, we 493 found that macropinocytosis continues at a high rate in mutants of the PKA 494 catalytic subunit, possibly accounting for the strong chemotactic defect of these 495 strains(Scavello et al, 2017). This could also be a confounding issue for some 496 other strains with early developmental defects(Khosla et al., 2005, Wu et al, 497 1995, Rodriguez et al, 2008, Lee et al, 2005).…”

The past, present and future of Dictyostelium as a model system

“…Sensitivity to chemoattractants appears to be negatively and positively controlled by PKA, HECT ubiquitin ligase-dependent ubiquitination and a novel negative regulator of Ras signalling (Scavello et al, 2017;Pergolizzi et al, 2017;Xu et al, 2017), whereas stochastic as well as chemotactically oriented pseudopod formation could be driven by a coupled excitable Ras/PI(3-4)P2/F actin system (van Haastert et al, 2017;Li et al, 2018).…”