FIGURE 7.

An equivalent mutation to that which rendered active Mpk1, Erk1, and Erk2 also rendered active the Drosophila Rolled. The indicated Rolled mutants, purified from E. coli, were treated (+MEK1) or not treated (–MEK1) with active MEK1 and ATP. Then, they were subjected to a standard kinase assay with MBP as a substrate. A, a fixed volume from each reaction was subjected to SDS-PAGE. Coomassie Brilliant Blue staining verified that equal amounts of substrate were loaded (lower panel). Then, the gel was exposed to x-ray film (upper panel). Note that the Rolled^R80S and the Rolled^R80S+D334N variants are active independently of MEK1 phosphorylation. B, using the paper-spotted kinase assay technique, we quantified and normalized the activities of the mutants to that of the MEK1-activated Rolled^WT that was defined as 100%. The Rolled^R80S exhibited 30% activity relative to that of MEK1-activated Rolled^WT. The Rolled^R80S/D334N exhibited 75% activity relative to that of the MEK1-activated Rolled^WT. Results shown are the average of two independent experiments, each performed in triplicates.