TPCK inhibits AGC kinases by direct activation loop adduction at phenylalanine-directed cysteine residues

► TPCK inhibits all tested AGC kinases with phenylalanine-directed, cysteine-containing activation loops, including RSK1, Akt1, S6K1, MSK1 and MSK2. ► Mass spectrometry identified TPCK adduction to a cysteine residue in the activation loop of RSK1, Akt1 and S6K1. ► Cysteine to alanine mutations within the activation loops of RSK1 and MSK1 lead to kinases partially resistant to TPCK. ► A Phenylalanine to Leucine mutation in the activation loop of RSK1 leads to partial TPCK resistance.