ERK5 (extracellular-signal-regulated kinase 5), also termed BMK1 [big MAPK1 (mitogen-activated protein kinase 1)], is the most recently discovered member of the MAPK family. It is expressed in a variety of tissues and is activated by a range of growth factors, cytokines and cellular stresses. Targeted deletion of Erk5 in mice has revealed that the ERK5 signalling cascade is critical for normal cardiovascular development and vascular integrity. In vitro studies have revealed that in endothelial cells, ERK5 is required for preventing apoptosis, mediating shear-stress signalling, regulating hypoxia, tumour angiogenesis and cell migration. This review focuses on our current understanding of the role of ERK5 in regulating endothelial cell function.
- big mitogen-activated protein kinase 1 (BMK1)
- endothelial cell
- extracellular-signal-regulated kinase 5 (ERK5)
- mitogen-activated protein kinase (MAPK)
- signal transduction
MAPKs (mitogen-activated protein kinases) play an essential role in regulating many cellular processes including growth, differentiation and apoptosis. MAPKs are activated by a range of growth factors and chemical stimuli such as oxidative stress and osmotic imbalance, and are responsible for transducing extracellular signals to the cytoplasm and nucleus. In mammalian cells, the MAPK signalling system consists of four distinct linear signalling cascades terminating in ERK1/2 (extracellular-signal-regulated kinase 1 and 2), JNK1–3 (c-Jun N-terminal kinases 1, 2 and 3), p38 MAPKs (p38 α, β, γ and δ) or the most recently discovered MAPK, ERK5 [1,2]. Each of these terminal kinases phosphorylates a variety of cellular targets ranging from cytoplasmic enzymes to transcription factors .
Identification of ERK5
ERK5 was cloned by two independent research groups in 1995. Dixon and co-workers first identified MEK5 and then utilized a yeast two-hybrid assay to identify binding partners, resulting in the discovery of ERK5 . In a separate study, Lee et al.  used degenerate PCR to screen a human placenta cDNA library and isolated a novel MAPK, which, due to its relatively large size when compared with ERK1 and ERK2, they termed BMK1 (big MAPK1). It later became apparent that BMK1 and ERK5 were in fact the same protein. In vertebrates, ERK5 is expressed in a variety of tissues, showing high abundance in heart, brain, lung, skeletal muscle, placenta and kidneys [4,5]. ERK5 is also widely expressed in a number of different cell lines .
Structure of ERK5
The human ERK5 gene (also termed MAPK7) is present on chromosome 17p11.2 and spans 5.79 kb. It has an open reading frame of 2445 bp encoding a protein of 816 amino acids with a predicted molecular mass of 98 kDa (Figure 1). ERK5 shares 66% sequence homology with ERK1/2 within the kinase domain, which contains the TEY dual phosphorylation motif in the activation loop . The N-terminal domain of ERK5 contains the kinase domain (amino acids 78–406). In addition, it is important for cytoplasmic targeting (amino acids 1–77), interaction with MEK5 (amino acids 78–139) and oligomerization (amino acids 140–406) [7,8]. The large size of ERK5 is attributable to its long C-terminal tail of approx. 400 amino acids, which is unique among the MAPKs. The C-terminal domain contains an NLS (nuclear localization signal) (amino acids 505–539) and two proline-rich domains (amino acids 434–465 and 578–701) that are proposed to serve as binding sites for SH3 (Src homology 3)-domain-containing proteins [4,8]. The C-terminal region also contains a MEF2 (myocyte enhancer factor 2)-interacting region (amino acids 440–501) and a transcriptional activation domain (amino acids 664–789) that regulates MEF2 transcription factor activity . Truncation of the C-terminal tail results in increased ERK5 kinase activity, revealing that the C-terminal tail of ERK5 has an autoinhibitory function .
Activation of the ERK5 signalling axis
ERK5 was originally identified as a stress-activated MAPK, activated by both osmotic and oxidative stresses . Subsequent studies have revealed that it is also activated by serum  and a range of growth factors including EGF (epidermal growth factor) , FGF-2 (fibroblast growth factor-2)  and VEGF (vascular endothelial growth factor)  and by cytokines such as LIF (leukaemia inhibitory factor)  and IL-6 (interleukin 6)  (Figure 2). ERK5 is also activated by a range of physiological and pathological conditions such as fluid shear stress , hypoxia  and ischaemia .
Activation of a MAPK signalling module consists of the initial activation of a MAPKKK (MAPKK kinase), resulting in the sequential activation of MAPKK and ultimately MAPK  (Figure 2). MEKK2 (MAP/ERK kinase kinase 2) and MEKK3 phosphorylate MEK5 on Ser311 and Thr315, resulting in an increase in MEK5 activity [9,19]. ERK5 contains a dual phosphorylation motif (TEY) in its activation loop and is phosphorylated on Thr218/Tyr220 by the upstream kinase MEK5, resulting in an increase in the catalytic activity of ERK5 [4,5,20]. MEK5 preferentially phosphorylates ERK5 on Thr218, which is believed to induce a conformational change facilitating the subsequent phosphorylation of Tyr220 leading to full catalytic activity . Active ERK5 is able to undergo autophosphorylation on a number of residues and can also phosphorylate MEK5 . A recent study has identified a number of residues within the C-terminal tail of ERK5, which are autophosphorylated, leading to an enhancement of ERK5 transcriptional activity .
Dephosphorylation of MAPKs on the TXY motif by an MKP (MAP kinase phosphatase) subfamily of DUSPs (dual-specificity phosphatases) leads to their inactivation . Currently, no DUSP has been identified that dephosphorylates ERK5. However, ERK5 is dephosphorylated by the phosphotyrosine-specific phosphatase PTP-SL (protein tyrosine phosphatase STEP-like), which interacts with ERK5 and impedes its translocation to the nucleus . ERK5 is also regulated by other post-translational modifications in addition to phosphorylation. It has recently been reported that ERK5 undergoes SUMOylation by SUMO3 (small ubiquitin-related modifier 3) on Lys6 and Lys22 after treatment with H2O2 and AGEs (advanced glycation end-products) in HUVECs (human umbilical vein endothelial cells) .
Similar to other MAPKs, ERK5 belongs to a family of evolutionarily conserved proline-directed protein kinases that phosphorylate substrates on serine and threonine residues immediately preceding a proline residue. However, certain serine and threonine autophosphorylation sites in ERK5 are not followed by proline [21,22], suggesting that the specificity of ERK5 may differ from other MAPK family members. Activation of the ERK5 signalling axis stimulates both distinct and similar pathways to the classical ERK1/2 pathway . Downstream targets of ERK5 include the MEF2 transcription factor family members MEF2A, MEF2C and MEF2D [9,27,28]. Other targets include the Ets domain transcription factor Sap1a , c-Myc  and CREB (cAMP-response-element-binding protein) .
Role of ERK5 in vivo
To address the physiological role of the ERK5 signalling axis, researchers have utilized gene targeting in mice to ablate specific genes (Table 1; ). Erk5-deficient mice die at approximately E10.5 (embryonic day 10.5) due to cardiovascular defects and angiogenic failure in embryonic and extraembryonic tissues. In these mice, the developing vasculature fails to mature, with endothelial cells becoming disorganized and rounded, leading to a loss of vascular integrity [16,33,34]. Similar phenotypic abnormalities are seen in mice lacking Mek5  and Mekk3 , suggesting that the ERK5 signalling axis is critical to vasculogenesis and angiogenesis. In an attempt to determine the primary defect on ERK5 gene ablation, researchers have generated conditional tissue-specific ERK5-knockout mice. Endothelial-specific Erk5-knockout mice show cardiovascular defects and die at approx. E10.0, similar to the conventional Erk5-knockout mice . However, knockout of Erk5 specifically in cardiomyocytes does not affect development . These important data suggest that whereas global Erk5 knockout affects cardiovascular development, the initial defect occurs in the endothelium and that ERK5 is critical for endothelial cell function. The requirement of ERK5 in the maintenance of vascular integrity is highlighted by the fact that induced ablation of Erk5 in adult mice is lethal within 2–3 weeks as blood vessels become leaky due to endothelial cell apoptosis .
ERK5 and endothelial cell physiology
Inhibition of endothelial apoptosis
Targeted deletion of the ERK5 signalling axis in mice suggests that ERK5 plays an essential role in endothelial cell physiology (Table 1). Initial studies using HUVECs stimulated with H2O2 showed that ERK5 was a redox-sensitive kinase . Further studies demonstrated that flow-induced shear stress and osmotic stress could stimulate ERK5 activity in BAECs (bovine aortic endothelial cells) . Given the known atheroprotective effects of laminar flow , Pi et al.  subsequently demonstrated that ERK5 is required for mediating flow-stimulated survival in BLMECs (bovine lung microvascular endothelial cells). This study  revealed that ERK5 induced the phosphorylation and inactivation of the pro-apoptotic protein Bad at Ser112 and Ser136, thus sequestering Bad in the cytoplasm and preventing the subsequent activation of caspase 3 and cell death . Bad does not contain a MAPK consensus sequence, suggesting that ERK5 does not phosphorylate Bad directly. Surprisingly, other candidate kinases such as Akt/PKB (protein kinase B), PKA (protein kinase A) and p90RSK (RSK is ribosomal S6 kinase), which are known to phosphorylate Bad, were not responsible for mediating ERK5-induced phosphorylation of Bad in these cells . However, recent results have shown that in murine fibroblasts, ERK5 protects against osmotic stress by inducing Akt phosphorylation on Ser473 and Thr308, leading to inactivation of the Foxo3a (forkhead box O3a) transcription factor and down-regulation of FasL expression .
Mediation of shear-stress signalling
Fluid shear-stress-mediated ERK5 activation has been shown to confer an atheroprotective effect by negatively-regulating TNFα (tumour necrosis factor α)-stimulated expression of adhesion molecules in endothelial cells . A more recent study utilizing a MEK5 inhibitor has revealed that the MEK5–ERK5 pathway mediates flow-dependent inhibition of TNFα signalling in BLMECs . Analysis of laminar shear-stress-induced transcriptional responses in endothelial cells has identified KLF2 (Krüppel-like factor 2) as a mechano-stress-induced gene [42,43]. KLF2 is responsible for negatively regulating inflammation and angiogenesis and maintaining vascular quiescence [43–45]. KLF2 has subsequently been identified as an ERK5 responsive gene in mouse embryonic fibroblasts in a pathway requiring MEF2 transcription factor . In addition, recent studies have shown that ERK5 is required for flow-induced expression of KLF2 in HUVECs , and the subsequent increased cell-surface expression of CD59 .
Regulation of hypoxia
ERK5 is activated under hypoxic conditions and has been reported to negatively regulate VEGF expression in mouse embryonic fibroblasts . Furthermore, increased VEGF expression was observed in the Erk5−/− embryos compared with wild-type and Erk+/− mice . VEGF is critical for vasculogenesis and angiogenesis . However, it is unlikely that increased VEGF is the primary defect leading to death in the Erk5−/− mice at E11 , as overexpression of Vegf in mouse embryos results in normal development up to E12.5, with lethality due to cardiovascular abnormalities only evident at E12.5–14.5 . ERK5 has been shown to regulate HIF-1α (hypoxia-inducible factor-1α) levels by promoting the ubiquitination and subsequent proteolysis of HIF-1α in BLMECs, leading to a decrease in hypoxia-induced VEGF mRNA levels . It has been shown that HIF-1α is directly phosphorylated by ERK1/2, leading to an increase in transcriptional activity [52,53]. It remains to be determined whether HIF-1α is also a direct substrate for phosphorylation by ERK5, possibly antagonizing activation by ERK1/2.
Angiogenesis is defined as the formation of new blood vessels from pre-existing vessels and plays a critical role in normal physiological development and in the pathology of diseases such as cancer . Hayashi et al.  have provided evidence that ERK5 regulates tumour angiogenesis. After the establishment of melanoma and Lewis lung carcinoma tumour xenografts in mice, induced ablation of Erk5 in Erk5flox/flox mice carrying an inducible Mx1-Cre transgene resulted in a regression of the tumour vasculature and a concomitant reduction in tumour volume by 63 and 72% respectively. Furthermore, screening of potential ERK5 targets using a Pepchip array revealed that ablation of Erk5 in mouse lung endothelial cells prevented phosphorylation of rpS6 (ribosomal protein S6) on Ser235/Ser236 by p90RSK . Interestingly, ablation of Erk5 in mouse fibroblasts did not affect phosphorylation of rpS6, suggesting that activation of this signalling pathway may be cell type specific.
The precise role of ERK5 in regulating VEGF-mediated angiogenesis still remains to be determined. It is known that both VEGF and FGF-2 stimulate ERK5 activity in HUVECs and MLCECs (mouse lung capillary endothelial cells) ; therefore it remains a distinct possibility that ERK5 is an important component of the VEGF signalling cascade in endothelial cells, responsible for regulating endothelial cell survival, permeability, proliferation and differentiation .
Recent results point to a role for the MEK5–ERK5 pathway in regulating endothelial cell migration and focal contact turnover . Expression of constitutively active MEK5 (MEK5DD, in which Ser313 and Thr317 are replaced by aspartate) led to hyperphosphorylation of FAK (focal adhesion kinase). Another recent report has implicated ERK5 in integrin-mediated cell adhesion and FAK phosphorylation in cancer cells . Taken together, these results suggest that ERK5 plays an important role in cell attachment to the extracellular matrix and cell migration.
Conclusions and perspectives
The ERK5 signalling axis was the most recent of the MAPK modules to be discovered. However, research over the past decade has revealed a vital role for this kinase cascade in normal physiology. Whereas ERK5 appears to be almost ubiquitously expressed in different tissues, the phenotype of the ERK5-knockout mice indicates that it is critical for endothelial cell physiology. However, conditional knockout in other cell types suggests a degree of redundancy with other signalling pathways . In vitro studies have revealed that ERK5 is important for endothelial and neuronal cell survival [58–60], suggesting that under certain conditions, these cell types have a critical dependence on ERK5 activity and may express specific ERK5 substrates not expressed in other cells. It is possible that certain diseases may be amenable to pharmacological intervention with modulators of the ERK5 signalling axis. Aberrant activation of ERK5 during tumour development in both the tumour  and vascular compartments  may present a therapeutic window for the use of ERK5 signalling inhibitors such as the recently developed MEK5 inhibitor . Conversely, stimulating ERK5 activity by gene therapy may offer a way of stimulating endothelial cell survival and revascularization under conditions such as ischaemia.
We acknowledge support from the Biotechnology and Biological Sciences Research Council/AstraZeneca through a CASE (Co-operative Awards in Science and Engineering) studentship (to O.Ll.R.) and the North West Cancer Research Fund (to K.H., J.M. and M.J.C.).
Molecular and Cellular Mechanisms of Angiogenesis: Biochemical Society Focused Meeting held at University of Chester, Chester, U.K., 15–17 July 2009. Organized and Edited by Ian Zachary (University College London, U.K.) and Sreenivasan Ponnambalam (Leeds, U.K.).
Abbreviations: BMK1, big mitogen-activated protein kinase 1; BLMEC, bovine lung microvascular endothelial cell; CREB, cAMP-response-element-binding protein; DUSP, dual-specificity phosphatase; E, embryonic day; ERK5, extracellular-signal-regulated kinase 5; FAK, focal adhesion kinase; FGF-2, fibroblast growth factor-2; HIF-1α, hypoxia-inducible factor-1α; HUVEC, human umbilical vein endothelial cell; KLF2, Krüppel-like factor 2; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; MEF2, myocyte enhancer factor 2; MEK, MAP/ERK kinase; MEKK, MAP/ERK kinase kinase; NLS, nuclear localization signal; rpS6, ribosomal protein S6; RSK, ribosomal S6 kinase; p90RSK, p90 RSK; TNFα, tumour necrosis factor α; VEGF, vascular endothelial growth factor
- © The Authors Journal compilation © 2009 Biochemical Society