Identification and characterization of EppA as a potential substrate of DdERK2 in cAMP relay and chemotaxis of Dictyostelium

Abstract

The MAP kinase DdERK2 is critical for cAMP relay and chemotaxis to cAMP and folate, but the details downstream of DdERK2 are unclear. To search for targets of DdERK2 in Dictyostelium, 32PO 43--labeled protein samples from wild type and Dderk2- cells were resolved by 2 dimensional electrophoresis. Mass spectrometry was used to identify a novel 45kD protein as a substrate of DdERK2 in Dictyostelium. Mutation of potential DdERK2 phosphorylation sites demonstrated that phosphorylation on serine 250 of EppA is DdERK2-dependent. Changing serine 250 to alanine delayed development of Dictyostelium and impaired Dictyostelium chemotaxis to cAMP. Although overexpression of EppA had no significant effect on development or chemotaxis of Dictyostelium, disruption of the EppA gene led to delayed development and reduced chemotactic responses. Both EppA gene disruption and overexpression of EppA carrying the serine 250 to alanine mutation led to inhibition of intracellular cAMP accumulation in response to chemoattractant cAMP, a pivotal process in Dictyostelium chemotaxis and development. In addition to the deficiency in chemotaxis to cAMP, EppA disruptants also displayed deficiency in chemotaxis to folate. Our studies indicate that EppA regulates extracellular cAMP induced signal relay and chemotaxis of Dictyostelium in a DdERK2-dependent way.