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 has 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 cAMP stimulation and declines to basal levels after 5 minutes. In parallel with the DdERK2 activation is the appearance of a higher mobility band on Western blots. An antibody specific for activated MAP kinase shows that only the shifted band is tyrosine phosphorylated, suggesting that it is the active form. Both unstimulated and stimulated DdERK2 are soluble. In vitro phosphorylation with cell lysate supernatants or immunoprecipitates demonstrates the presence of several potential substrates, as identified by SDS-PAGE with mobility corresponding to molecular weights of 150, 25, and 19 kDa. Furthermore, immunoprecipitation studies suggest that these substrates are in a complex with DdERK2. These data suggest that DdERK2 works via cytoplasmic proteins to mediate signaling responses in Dictyostelium.
Wang
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Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Segall
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JE
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D002630:Chemotactic Factors / Q000502:physiology*; D000242:Cyclic AMP / Q000494:pharmacology; D003600:Cytosol / Q000502:physiology*; D004023:Dictyostelium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005492:Folic Acid / Q000494:pharmacology; D046911:Macromolecular Substances; D019950:Mitogen-Activated Protein Kinase 1; D010766:Phosphorylation; D011233:Precipitin Tests; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity
D002630:Chemotactic Factors; D046911:Macromolecular Substances; D005492:Folic Acid; D000242:Cyclic AMP; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1