JNK1β1 is phosphorylated during expression in E. coli and in vitro by MKK4 at three identical novel sites

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, JNK1β1, is further modified by MKK4 for phospho-regulation has not been previously investigated. Aiming to characterise the nature of JNK1β1 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 JNK1β1 in Escherichia coli, raising important questions that have significant implications for heterologous protein expression.

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► JNK1β1 is phosphorylated and becomes active during expression in E. coli.
► E. coli phosphorylated JNK1β1 can be completely dephosphorylated and deactivated.
► MEKK-C phospho-activated MKK4 can efficiently re-phosphorylate and activate JNK1β1.
► JNK1β1 is phosphorylated in E. coli and in vitro by MKK4 at the same 3 novel sites.