The role and regulation of VEGF-mediated ERK5 activity in endothelial cells

Nithianandarajah-Jones, Gopika The role and regulation of VEGF-mediated ERK5 activity in endothelial cells. PhD thesis, University of Liverpool.

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Abstract

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 physiological 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 phosphorylation of AKT in HDMECs. Furthermore, with an apparent co- localisation of ERK5 and AKT in the cytoplasm and at the plasma membrane of HDMECs, it was hypothesised that the two proteins were interacting partners. Adenoviral-mediated expression of FLAG-tagged ERK5 revealed that VEGFR-2, ERK5 and AKT co- immunoprecipitated in HDMECs, suggesting the possibility of a complex between these proteins at the cell periphery following VEGF stimulation and VEGFR-2 internalisation. Taken together, this study shows that VEGF appears to induce a unique activation of ERK5 in endothelial cells, which facilitates its interaction with AKT and subsequent regulation of AKT phosphorylation, ultimately regulating endothelial cell survival.

Item Type:	Thesis (PhD)
Additional Information:	Date: 2014-11 (completed)
Subjects:	?? Q1 ??
Depositing User:	Symplectic Admin
Date Deposited:	09 Sep 2015 15:13
Last Modified:	17 Dec 2022 01:37
DOI:	10.17638/02008164
URI:	https://livrepository.liverpool.ac.uk/id/eprint/2008164