Figure 2.

Role of the tail phosphorylation site in activation and phosphorylation of AGC kinases in vivo. COS7 cells were transfected with plasmid expressing haemagglutinin (HA)- or glutathione S-transferase (GST)-tagged wt or mutant kinase. After 16 h and a final 4 h serum-starvation period, cells were exposed to 1 μM insulin for 10 min (PKBα), to 20 nM EGF for 30 min (S6K1) or 15 min (RSK2), to 10 μg/ml anisomycin for 40 min (MSK1) or left in serum-containing medium (PRK2_Δ1−500) and then lysed. The kinases were precipitated from aliquots of the cell lysates with antibody to the HA tag or with glutathione beads. The precipitates were subjected to kinase assay, to immunoblotting with the indicated phosphorylation site-specific Ab or anti-HA Ab or stained for protein. Experiments were repeated at least three times and activity data (expressed as percent) are means±s.d.