Function and specificity in signaling pathway of two map kinases ERK1 and ERK2 in Dictyostelium

Abstract

Scope and Method of Study: MAPKs are associated with several human diseases. However, studying MAPK signaling pathways in higher eukaryotes is challenging due to presence of several MAPKs. Dictyostelium, whose genome encodes only two MAPKs ERK1 and ERK2, therefore offers many advantages in this respect. Our purpose in this research is to define the function and the specificity in signaling pathway of ERK1 and ERK2 through characterizing G protein-mediated pathways and searching for ERK-associated proteins. We used immunoprecipitation, pull-down assays and genetic analyses to examine the interactions of ERKs with wild-type and mutated Ga subunits. We also created ERK mutants and performed the complementation, suppression and epistasis tests to determine role of ERKs in Ga subunit-mediated pathways. To search for ERK-associated proteins, His6-tagged ERK1 and ERK2 complexes were isolated and then subjected to mass spectrometry analysis. Cellular distribution of the transcription factors (STATs), expression of the cell type-specific genes (ecmA, ecmB) and the knockout of the phosphodiesterase RegA were used to determine the involvement of these ERK-associated proteins in ERK pathways.

Findings and Conclusions: Our results showed that the Ga4 subunit interacts with ERK2 and the Ga5 subunit associates with ERK1 through the D-motifs. The Ga4-ERK2 interaction contributes to spore development while the Ga5-ERK1 interaction regulates cell viability and tip morphogenesis. We also indicated that ERK1 and ERK2 have different roles in Ga subunit-mediated pathways. Genetic analyses of the ERK-associated proteins revealed that both ERK1 and ERK2 negatively regulate RegA; the Ga5 subunit-ERK1 pathway controls nuclear localization of STATc ultimately resulting in repression of ecmA while the Ga2/Ga4 subunits-ERK2 pathways regulate function of STATa and expression of ecmB through down regulating RegA.