Discovery of a gatekeeper residue in the C terminal tail of the extracellular signal-regulated protein kinase 5 (ERK5)

The extracellular signal-regulated protein kinase 5 (ERK5) is a non-redundant mitogen-activated protein kinase (MAPK) that exhibits a unique C terminal extension which comprises distinct structural and functional properties. Here, we sought to elucidate the significance of phosphoacceptor sites in the C terminal transactivation domain of ERK5. We have found that Thr732 acted as a functional gatekeeper residue controlling C terminal-mediated nuclear translocation and transcriptional enhancement. Consistently, using a non-bias quantitative mass spectrometry approach, we demonstrated that phosphorylation at Thr732 conferred selectivity for binding interactions of ERK5 with proteins related to chromatin and RNA biology, whereas a number of metabolic regulators were associated with full-length wild type ERK5. Additionally, our proteomic analysis revealed that phosphorylation of the Ser730-Glu-Thr732-Pro motif could occur independently of dual phosphorylation at Thr218-Glu-Tyr220 in the activation loop. Together these results firmly establish the significance of C terminal phosphorylation in regulating ERK5 function, independently of MEK5. This novel mechanism may be of particular relevance in cancer cells where ERK5 has be found to be hyperphosphoryated on its C terminal tail.