EppA, a putative substrate of DdERK2, regulates cyclic AMP relay and chemotaxis in Dictyostelium discoideum.
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, 32PO4(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-to-alanine 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.
Chen
Songyang
S
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, New York 10461, USA.
Segall
Jeffrey E
JE
D000595:Amino Acid Sequence; D000818:Animals; D002633:Chemotaxis / Q000235:genetics*; D000242:Cyclic AMP / Q000378:metabolism*; D004023:Dictyostelium / Q000254:growth & development / Q000502:physiology*; D015180:Electrophoresis, Gel, Two-Dimensional; D018390:Gene Targeting; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology*; D040901:Proteomics; D017384:Sequence Deletion; D015398:Signal Transduction
D010750:Phosphoproteins; D000242:Cyclic AMP; D019950:Mitogen-Activated Protein Kinase 1