Role of p38-MAPK Signaling in Cell Kinase Activities

1. mkk3DN mkk6DN control – + SB202190 –/– –/– +/– +/– mkk7 – – – UV CycB1 k.a. GST-ATF2 CDC2 GST-c-Jun CycD1 k.a. Actin Mkk6DN Cdk4 MKK3DN + + + a Supplementary Fig. 1 b (a) Immune-complex kinase assays of p38-MAPK and JNK using GST-ATF2 and GST-c-Jun as substrates, respectively, in the presence of MKK3-DN and MKK6-DN. mkk7+/- MEFs were used. Actin blots from total cell lysates are shown as control. Ectopically expressed MKK3DN and MKK6DN, which carry HA-tag at N-terminus, were confirmed using anti-HA antibody. Controls are mkk7+/- cells transfected with an empty pBabe vector. (b)Inhibition of p38-MAPK by SB202190 (10 mM) enhances CyclinB1 associated kinase activities (k.a.) even in mkk7+/- MEFs. Note that CyclinB1, but not CyclinD1 associated kinase activities are enhanced by inhibition of p38-MAPK (passage 2-3). Expression of Cdk4 and CDC2 was determined by Western blot.

2. a (h) after hepatectomy 0 12 24 48 Supplementary Fig. 2 + + + + Dliv Dliv Dliv Dliv Jun + + Dliv Dliv Dliv Dliv p38-MAPKa + + CycB1/CDC2 k.a. CycB1 CDC2 b (h) after hepatectomy 0 48 Dliv Dliv + Jun + Dliv Dliv + + p38-MAPKa CycB1 k.a. CycB1 (a) Genetic deletion of p38-MAPKa restores reduced CyclinB1 associated kinase activities (k.a.) in jun deficient livers following partial hepatectomy. Kinase activities were determined at the indicated time points. Protein expression levels of CyclinB1 and Cdc2 were determined by Western blot. (b)Genetic deletion of p38-MAPKa restores reduced CyclinB1 associated kinase activities (k.a.) in jun deficient livers at 48 hours after partial hepatectomy. Kinase activities were determined at 48 hours after partial hepatectomy. Protein expression levels of CyclinB1 were determined by Western blot.