EppA, a Putative Substrate of DdERK2, Regulates Cyclic AMP Relay and Chemotaxis in Dictyostelium discoideum

Abstract

The mitogen-activated protein kinase DdERK2 is critical for cyclic AMP (cAMP) relay and chemotaxis to cAMP and folate, but the details downstream of DdERK2 are unclear. To search for targets of DdERK2 in Dictyostelium discoideum, 32 PO 4 3؊ -labeled protein samples from wild-type and Dderk2 ؊ cells were resolved by 2-dimensional electrophoresis. Mass spectrometry was used to identify a novel 45-kDa protein, named EppA (ERK2-dependent phosphoprotein A), 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 reduced Dictyostelium chemotaxis to cAMP. Although overexpression of EppA had no significant effect on the development or chemotaxis of Dictyostelium, disruption of the eppA gene led to delayed development and reduced chemotactic responses to both cAMP and folate. Both eppA gene disruption and overexpression of EppA carrying the serine 250-toalanine mutation led to inhibition of intracellular cAMP accumulation in response to chemoattractant cAMP, a pivotal process in Dictyostelium chemotaxis and development. Our studies indicate that EppA regulates extracellular cAMP-induced signal relay and chemotaxis of Dictyostelium.

Paxillin and Phospholipase D Interact To Regulate Actin-Based Processes in Dictyostelium discoideum

“…In this assay, cells move up a stable chemotactic gradient by crawling underneath a thin layer of agarose. Wild-type cells moved toward the cAMP source with an average speed of 7.9 Ϯ 0.4 m/min, which is consistent with previous reports (9,47,55). The speed of paxB Ϫ cells was increased 80% over that of wild-type cells.…”

“…Thus, cells that move in a straight line have the maximal directionality value of 1, while cells that make a random walk and end up back at their starting point have a directionality of 0. In accordance with previous work, wild-type cells moved with a directionality of 0.65 Ϯ 0.05 (9,55). For paxB Ϫ cells, the directionality was similar to that of wild-type cells.…”

Absence of catalytic domain in a putative protein kinase C (PkcA) suppresses tip dominance in Dictyostelium discoideum

“…Cell movement towards cAMP was recorded for 15 min at 30 s intervals, and the average velocity (total displacement divided by time) was calculated. In agreement with earlier reports, the velocity of Ax2 cells was 10.04±1.24 μm/min (Chen and Segall, 2006; Plak et al ., 2013; Veltman et al ., 2008), and interestingly, that of pkcA − cells was 13% higher (Fig. 5) The chemotaxis defects of pkcA − was restored in the complemented strain pkcA − /[act15: pkcA ] with responses similar to Ax2.…”

TheDictyosteliumprestalk inducer differentiation-inducing factor-1 (DIF-1) triggers unexpectedly complex global phosphorylation changes

“…EppA is an ERK2 substrate and is phosphorylated in response to cAMP; like ERK2, it has a role in cAMP relay and chemotaxis ( Chen and Segall, 2006 ). We found a more than fivefold dephosphorylation of EppA (S325) after 5 min of DIF-1 treatment ( Figure 7A ).…”