JNK1 is activated by phosphorylation of the canonical T183 and Y185 residues, modifications that are catalysed typically by the upstream eukaryotic kinases MKK4 and MKK7. Nonetheless, the exact sites at which the most abundant JNK variant, JNK1 beta, is further modified by MKK4 for phospho-regulation has not been previously investigated. Aiming to characterise the nature of JNK1 beta phosphorylation by active MKK4 using mass spectrometry, a recognised yet uncharacterised phospho-site (S377) as well as two novel phospho-residues (T228 and S284) were identified. Interestingly, the identical sites were phosphorylated during overexpression of JNK1 beta 1 in Escherichia coli, raising important questions that have significant implications for heterologous protein expression. (C) 2013 Elsevier Inc. All rights reserved.