Abstract
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. Collectively, our results firmly establish the significance of C-terminal phosphorylation in regulating ERK5 function. The post-translational modification of ERK5 on its C-terminal tail might be of particular relevance in cancer cells where ERK5 has be found to be hyperphosphoryated.
Highlights
Mitogen-activated protein kinase (MAPK) pathways are evolutionarily conserved signaling cascades involved in the regulation of a variety of fundamental cellular processes such as cell proliferation, differentiation, survival and migration [1]
We propose that single phosphorylation at Thr732 stabilizes extracellular signal-regulated protein kinase 5 (ERK5) in an open conformation where the NLS is exposed to facilitate nuclear entry
This is functionally important given that Thr732 by alanine (T732A) mutation blocked the nuclear import of ERK5 caused by activating phosphorylation of ERK5 by MAPK/ERK kinase 5 (MEK5) in response to epidermal growth factor (EGF) stimulation
Summary
Mitogen-activated protein kinase (MAPK) pathways are evolutionarily conserved signaling cascades involved in the regulation of a variety of fundamental cellular processes such as cell proliferation, differentiation, survival and migration [1]. These pathways comprise a three-tier protein kinase cascade which results from the sequential activation of a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPK) and a MAPK, to relay, amplify and integrate many different stimuli, including mitogens, cytokines and various environmental stresses. The C-terminal tail of ERK5 contains two proline-rich domains (PR1 (aa 434-464) and PR2 (aa 578-701)), a myocyte enhancer-binding factor 2 (MEF2)-interacting domain (aa 440-501), a nuclear localization signal (NLS (aa 505-539)) and a transactivation domain (TAD (aa 664-789)) [5]
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