TY - JOUR
T1 - The dictyostelium MAP kinase DdERK2 functions as a cytosolic protein in complexes with its potential substrates in chemotactic signal transduction
AU - Wang, Yiwen
AU - Segall, Jeffrey E.
N1 - Funding Information:
We thank the members of the Segall and Condeelis labs for many helpful discussions and Ying Ping Yu for technical assistance. J. Segall is an Established Scientist of the New York City Af®liate of the American Heart Association. This research was supported by NIHGM44246. A portion of this work was presented at the American Society for Cell Biology (Mol. Biol. Cell 6, 127a). The data in this paper will be submitted in partial ful®llment of the requirement for the Degree of Doctor of Philosophy in the Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University.
PY - 1998/3/6
Y1 - 1998/3/6
N2 - 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.
AB - 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.
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U2 - 10.1006/bbrc.1997.8118
DO - 10.1006/bbrc.1997.8118
M3 - Article
C2 - 9514860
AN - SCOPUS:0032489287
SN - 0006-291X
VL - 244
SP - 149
EP - 155
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 1
ER -