The strong activation of ERK5 in HeLa seen upon EGF stimulation (Figure 3.1a, Figure 3.2a and Figure 3.5) corroborates findings from previous studies although these include a variety of cell types (Kato et al., 1998; Kamakura et al., 1999; Hayashi et al., 2004; Kondoh et al., 2006). With the use of conventional SDS-PAGE, a phospho-specific ERK5 antibody against the Thr218/Tyr220 residues and Phos-tag™ SDS-PAGE, it was possible to ascertain that in HeLa cells, ERK5 not only undergoes phosphorylation in the activation loop of the kinase domain (Figure 3.2a), but also at additional residues. As a consequence of this extensive phosphorylation, ERK5 was able to produce a mobility bandshift with conventional SDS-PAGE (Figure 3.1a), as well as a dual bandshift (p-ERK5 and hyper p- ERK5) with Phos-tag™ SDS-PAGE (Figure 3.5) in the HeLa cells.
TGF-α possesses some structural similarity to EGF and is able to bind to the EGF receptor (Tam et al., 1984; Schreiber et al., 1986) with an affinity indistinguishable from EGF in an equilibrium competition assay (Ebner and Derynck, 1991), consequently potentiating the activation of ERK5 in HeLa cells through EGFR-1. In addition to this, it has recently been shown that although the HGF receptor (cMET) is usually expressed in cells of an epithelial origin, they are also expressed in HeLa cells and dimerise upon ligand binding (Dietz et al., 2013). The number of cMET receptors on the surface of HeLa cells is significantly lower than that of the EGF receptor (4600-8700 cMET receptors/HeLa (Dietz et al., 2013) compared to 22,000 high affinity EGFR/HeLa and 25,000 low affinity EGFR/HeLa (Berkers et al., 1991)). Thus, the activation of ERK5 upon HGF stimulation is less than that of EGF, which can be seen by the presence of a less intense phosphorylation band for HGF compared to that of EGF (Figure 3.1a, Figure 3.2a and Figure 3.5). Furthermore, the Phos- tag™ SDS-PAGE revealed that although TGF-α may bind to EGFR-1 with a similar affinity as EGF, the former agonist did not appear to phosphorylate ERK5 to the same extent as EGF, highlighting the potency of EGF-mediated ERK5 activation.
In stark contrast to ERK5 activation in HeLa cells, it was impossible to detect ERK5 activation in HDMEC using the mobility bandshift method with conventional SDS-PAGE (Figure 3.1b). However, the use of the p-Thr218/Tyr220 antibody and Phos-tag™ SDS-PAGE enabled the detection of ERK5 phosphorylation in HDMEC upon stimulation with VEGF (Figure 3.2b and Figure 3.6).
Chapter Three: Characterisation of ERK5 activation
Interestingly, this study revealed that both VEGF and HGF were able to stimulate AKT phosphorylation with much more potency than other agonists in HDMEC (Figure 3.6). It has previously been reported that in human malignant mesothelioma cells both AKT and ERK5 are activated by HGF stimulation (Altomare et al., 2005; Ramos-Nino et al., 2008), whereas in human aortic endothelial cells (HAEC), AKT and ERK1/2 are phosphorylated (Nakagami et al., 2001).
Studies have associated the cMET signalling pathway in tumour growth, survival, and metastasis; additionally promoting angiogenesis by acting synergistically with VEGF (van Belle et al., 1998; Xin et al., 2001). Furthermore, stimulation with VEGF has been reported to lead to increased tyrosine phosphorylation of PI3K and AKT (Guo et al., 1995).
Although there is evidence that FGF-2, HGF and EGF are able to phosphorylate PLCγ in other cell lines (Nishibe et al., 1990; Mohammadi et al., 1991; Okano et al., 1993), it was observed in this study that VEGF was unique in its ability to stimulate PLCγ amongst growth factors (Figure 3.6); a finding which has previously been observed in HDMEC (Holmes et al., 2010).
Utilisation of MAE cells stably expressing Flk-1 receptor mutants confirmed that the Tyr1173 residue (Tyr1175 in humans) was essential for PLCγ phosphorylation (Figure 3.8), which corroborates previous studies (Takahashi et al., 2001). Additionally, this residue was also responsible for the activation of ERK5, implying the presence of ERK5 downstream of VEGFR-2 (Tyr1175) and PLCγ (Figure 3.8). With the knowledge that p-PLCγ hydrolyses PIP2 into IP3 which increases intracellular Ca2+, and DAG an activator of PKC (Wu et al., 2000a), it is possible that ERK5 activity may be regulated by various PKC isoforms, which has previously been shown in a multitude of cell types (Diaz-Meco and Moscat, 2001; Li et al., 2005; Zhao et al., 2010). Contrary to these studies however, other groups have shown that ERK5 activation does not occur via PKC (Abe et al., 1996; Kato et al., 1998; Yan et al., 1999) suggesting that activation of this pathway is highly dependent on cell type.
Further to this, the MAE Tyr1173Phe mutant displayed reduced p-AKT, which may be attributed to decreased activation of PI3K in response to the hindered binding of Shb to the Tyr1173 residue (Holmqvist et al., 2004), whereas ERK1/2 activity remained unaffected demonstrating that ERK5 and ERK1/2 activity are independent of one another and regulated separately.
With the suggestions that VEGFR-2 and PLCγ are required for the VEGF-mediated phosphorylation of ERK5 in endothelial cells, attention turned to the apparent difference
Chapter Three: Characterisation of ERK5 activation
in ERK5 phosphorylation in HeLa cells namely, the hyper p-ERK5 band. The MAE WT cells enabled exploitation of the presence of both the VEGF and EGF cognate receptors on an endothelial cell type. Furthermore, utilising both Phos-tag™ and conventional SDS-PAGE allowed for discrimination of the different phosphorylation events of ERK5 amongst agonists and cell types (Figure 3.9).
This study revealed for the first time that observed differences in ERK5 phosphorylation were not dependent upon cell type, instead upon agonist. The data presented in this chapter show that VEGF is able to phosphorylate ERK5 at the very least on the Thr218/Tyr220 residues within the activation loop, resulting in a p-ERK5 band on Phos-tag™ SDS-PAGE (Figure 3.9). However, this phosphorylation event alone is not substantial enough to generate a mobility bandshift on conventional SDS-PAGE (Figure 3.9); a similar discovery was made in HEK293 cells (Mody et al., 2003). In contrast to this, EGF is able to phosphorylate ERK5 at Thr218/Tyr220 as well as additional residues, most probably in the C- terminal tail, and as a consequence gives rise to a hyper p-ERK5 band on Phos-tag™ SDS- PAGE and a mobility bandshift on conventional SDS-PAGE (Figure 3.9). An additional interesting discovery amongst these results is that of ERK5 phosphorylation upon VEGF and EGF co-stimulation; there is an apparent decrease in hyper p-ERK5 compared to that of EGF stimulation alone. One theory may be that VEGFR-2 and EGFR-1 activate different intracellular pools of ERK5, whereby VEGFR-2 sequesters ERK5 in an intracellular localisation that precludes it from being further phosphorylated by EGFR-1.
The data generated with the use of the small-molecule kinase inhibitors targeting MEK5 and ERK5, further demonstrated these VEGF- and EGF-mediated differences in ERK5 phosphorylation (Figure 3.10, Figure 3.11, Figure 3.12, and Figure 3.13). It was evident that inhibition of MEK5 kinase activity with BIX 02189 prevented dual-phosphorylation of ERK5 in both VEGF-stimulated HDMEC and EGF-stimulated HeLa (Figure 3.10 and Figure 3.11), as well as the subsequent phosphorylation of additional C-terminal residues in HeLa (Figure 3.11). XMD8-92 appears to be a type I, ATP-competitive inhibitor that affects the kinase activity of ERK5 i.e. hyper p-ERK5 in EGF-stimulated HeLa (Figure 3.12 and Figure 3.13) and not activation of the T-E-Y motif, a finding that has previously been reported by way of mobility band shift (Deng et al., 2011). Thus, data from this project provides evidence to support findings from previous studies that have demonstrated no effect on vasculature stability upon XMD8-92 treatment (Yang et al., 2010a). Further elucidation of VEGF and EGF-mediated differences in ERK5 phosphorylation and the effect this may have on its intracellular localisation, as well as on cell survival, is discussed in the next chapter.
Chapter Four: Differential regulation of ERK5 in HDMEC and HeLa