Extracellular signal-regulated kinase 5 (ERK5) is the newest member of the mitogen- activated protein kinase (MAPK) family following the discovery of ERK1/2, JNK and p38 MAPK. ERK5 consists of an N-terminal kinase domain, similar to the other MAPK members, and a large C-terminal domain, which is unique in structure and function from the other MAPK members. ERK5 is activated in response to a multitude of extracellular stimuli, including pro-angiogenic factors in endothelial cells. The

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... al importance of ERK5 was highlighted following Erk5 gene ablation in mice, where a severe disruption to cardiovascular development and loss of vascular integrity was observed, resulting in embryonic lethality. This study investigated the differences in ERK5 activation between vascular endothelial growth factor (VEGF) stimulation of primary human dermal microvascular endothelial cells (HDMECs) compared to epidermal growth factor (EGF) stimulation of HeLa (immortalised epithelial cervical cancer cell line) cells. It was discovered that in contrast to other growth factors, VEGF appeared unique in its ability to induce ERK5 phosphorylation in HDMECs, stimulating ERK5 activity via a VEGFR-2/PLCγ-dependent pathway. Utilisation of the innovative Phos-tagTM reagent in SDS-PAGE facilitated the novel discovery that VEGF was only able to induce phosphorylation of the threonine (Thr)218/tyrosine (Tyr)220 residues present within the activation loop of the kinase domain. In contrast, EGF stimulation of HeLa cells resulted in phosphorylation of ERK5 on Thr218/Tyr220 as well as additional C- terminal residues such as Thr732. It was further demonstrated that contrary to EGF, VEGF stimulation of HDMECs did not evoke a nuclear translocation of ERK5, instead, ERK5 appeared to localise to the cytoplasm and plasma membrane. The analysis of intracellular signalling pathways following treatment with small-molecule inhibitors against MAPK/ERK kinase 5 (MEK5) and ERK5 kinase activity, revealed that VEGF-mediated ERK5 activation regulated phosphory [...]