THE DICTYOSTELIUM MAP KINASE DDERK2 FUNCTIONS AS A CYTOSOLIC PROTEIN IN COMPLEXES WITH ITS POTENTIAL SUBSTRATES IN CHEMOTACTIC SIGNAL-TRANSDUCTION

A polyclonal antibody against a MAP kinase (DdERK2) in Dictyostelium h as been made and used to study DdERK2 activation and localization. The activation of DdERK2 by the chemoattractants cAMP and folate is rapid and transient. Its activity peaks between 15 and 60 seconds after cAM P stimulation and declines to basal levels after 5 minutes. In paralle l with the DdERK2 activation is the appearance of a higher mobility ba nd on Western blots. An antibody specific for activated MAP kinase sho ws that only the shifted band is tyrosine phosphorylated, suggesting t hat it is the active form. Both unstimulated and stimulated DdERK2 are soluble. In vitro phosphorylation with cell lysate supernatants or im munoprecipitates demonstrates the presence of several potential substr ates, as identified by SDS-PAGE with mobility corresponding to molecul ar weights of 150, 25, and 19 kDa. Furthermore, immunoprecipitation st udies suggest that these substrates are in a complex with DdERK2. Thes e data suggest that DdERK2 works via cytoplasmic proteins to mediate s ignaling responses in Dictyostelium. (C) 1998 Academic Press.