FIGURE 4.
S6KII kinase activity and ERK-binding are required for phosphorylation of the protein. A, blot containing S6KII K231R, R902A, and K231R/R902A protein, probed with antibodies against mammalian pS380 RSK (fly pS515 S6KII), mammalian pS221 RSK (fly pS357 S6KII) and N-cadherin as a loading control. A separate blot with S6KII K597M protein was similarly probed. A third blot with proteins from all domain mutants was probed with an antibody against mammalian pT732 RSK (fly pT573 S6KII). Variations in the amount of total phosphorylation on the S6KII protein cause the mutants to have different electrophoretic mobilities. Transgene abbreviations: K231R (KR), R902A (RA), K231R/R902A (KR/RA), K597M (Km). B, blots containing the phosphorylation-site mutant proteins were probed sequentially with all three phospho-specific antibodies and anti-N-cadherin. The first three lanes (+, TA, TE) are imaged from one blot while the last three lanes (+, SA, SD) are all from a second blot. Transgene abbreviations: S515A (SA), S515D (SD), T732A (TA), T732E (TE). timUG4 indicates the timUAS-Gal4 driver. Histograms in A and B represent phospho signal normalized to protein abundance for the S6KII isoform (mean ± range). Numbers below the histograms in panels A and B correspond to the numbered lanes on the blots above. A star (*) represents a significant difference versus S6KII+. RA, KRRA (pS515), p = 0.02; KM (pS515), p = 0.008; SA, S.D. (pS515), p = 0.001; KM (pS357), p = 0.03); SA, S.D. (pS357), p = 0.01; RA, KRRA, KM, TA, TE (pT732), p = 0.001. All blots are representative of at least two independent experiments that yielded similar results.