Extracellular signal-re gulated kinase 5 (ERK5, also named big MAP kinase 1, Bmk1) is a member of the Mitogen-activated protein kinases (MAPKs). ERK5 is ubiquitously expressed throughout all mammalian tissues and cell lines (Lee et al., 1995; Zhou et al., 1995; Buschbeck and Ullrich, 2005), where it is activated in response to several growth factors and oxidative and hyperosmotic stress (Kato et al., 2000). ERK5 is twice the size of the classical MAPKs (816 aa for human ERK5), containing an N-terminal kinase domain (aa 78–406) and a unique C-terminal tail (aa 410–816) with no homology to any other protein, which has an autoinhibitory function (Buschbeck and Ullrich, 2005). This C-terminal tail contains a myocyte enhancer factor 2 MEF2-interacting region (aa 440–501, Kasler et al., 2000), a bipartite nuclear localization signal NLS (aa 505–539), and a transcriptional activation domain (TAD, aa 664–789, Kasler et al., 2000), which associates with and activates several transcription factors (Morimoto et al., 2007; Woo et al., 2010). Activation of ERK5 requires dual phosphorylation of a Threonine and Tyrosine residues within a TEY motif in the activation loop of the kinase domain (Mody et al., 2003). MEK5 is the only kinase that activates ERK5, and it has a unique specificity for ERK5, not phosphorylating any other MAPKs: MEK5 knockout mice dye at early stages of embryo development, showing similar defects in cardiac development and angiogenesis as those reported for ERK5 knockout mice (Wang et al., 2005).

ERK5 plays a crucial role in cell proliferation. First evidences reported that ERK5 activation is required for EGF-dependent proliferation in HeLa cells (Kato et al., 1998), by inducing transcription of c-jun (an essential component in cell proliferation) through the transcriptional activation of MEF2C (Kato et al., 1997). Since then, several authors have shown activation of ERK5 in response to other mitogenic factors, such as Nerve growth factor (NGF, Shao et al., 2002), Granulocyte colony-stimulating factor (G-CSF, Dong et al., 2001), Fibroblast growth factor (FGF, Kesavan et al., 2004), or Platelet-derived growth factor (PDGF, Rovida et al., 2008).

ERK5 regulates cell cycle progression, being necessary for G1/S transition. In this regard, ERK5 inhibition prevents cells from entering the S phase of the cell cycle (Kato et al., 1998) by stabilizing the cyclin-dependent protein kinase (CDK) inhibitors p21 and p27 (Perez-Madrigal et al., 2012). In human breast cancer MDA-MB-231 cells, activation of ERK5 promotes c-Myc-dependent transcriptional activation of miR-17-5p and miR-20a, resulting in blockade of p21 mRNA translation (Perez-Madrigal et al., 2012). ERK5 also mediates in G1/S transition by regulating expression of cyclin D1. Activation of MEK5/ERK5 pathway induces transcription of Cyclin D1, resulting in cell cycle progression in G1. Conversely, ERK5 inhibition diminishes serum-induced Cyclin D1 protein levels (Mulloy et al., 2003). Additionally, ERK5 is also implicated in G2/M transition. ERK5 is activated at G2/M, it is required for timely mitotic entry, and constitutively active ERK5 increases the mitotic index (Cude et al., 2007; Girio et al., 2007). The mitotic entry induced by ERK5 depends on the activation of the transcription factor NF-kB, which upregulates mitosis-promoting genes such as cyclins B1 and B2, and cdc25B (Cude et al., 2007). During mitosis, active ERK5 prevents caspase activation by binding and inactivating the pro-apoptotic protein Bim, suggesting that active ERK5 contributes to cell survival in mitosis (Girio et al., 2007). The role of ERK5 in controlling cell survival and differentiation, as well as angiogenesis, has been already covered in excellent reviews (Wang and Tournier, 2006; Drew et al., 2012; Lochhead et al., 2012; Nithianandarajah-Jones et al., 2012).

During the last years, different laboratories have shown that the MEK5-ERK5 pathway plays a key role in cancer cell proliferation. For instance, overexpression of either MEK5 or ERK5 in prostate adenocarcinoma PC-3 cells results in increased proliferation index (McCracken et al., 2008; Erazo et al., 2013). Consequently, ERK5 kinase inhibitors (such as the XMD8-92 compound) or ERK5 silencing show antiproliferative activity in different cancer cell lines and block tumor growth in animal models (human tumor xenografts). Table 1 summarizes the different human cancers where it has been reported an effect of ERK5 silencing/inhibition on cell proliferation and/or tumor growth. Importantly, there are increasing evidences pointing to an important role of nuclear ERK5 in cancer, both in vitro (cell lines) and in vivo (mouse models). For instance, there is a strong correlation between nuclear ERK5 and poor prognosis in prostate cancer patients. Expression of nuclear ERK5 is upregulated in prostate cancers showing high-grade Gleason and bone metastasis (McCracken et al., 2008; Clape et al., 2009; Ramsay et al., 2011; Ahmad et al., 2013). However, and as explained below, ERK5 can promote cell proliferation independently of its kinase activity, acting as a moonlighting protein. For instance, hepatocellular carcinoma (HCC) tumors show increased nuclear ERK5, which does not correlate with an increase on ERK5 kinase activity (Rovida et al., 2015). Also, ERK5 localizes at the nucleus of CLB-BAR and CLB-GE human neuroblastoma cell lines, even in the presence of the ERK5 inhibitor XMD8-92 (Umapathy et al., 2014). These findings suggest that nuclear ERK5 expression, instead of ERK5 phosphorylation, might be used as prognostic biomarker of some cancers.

The best characterized ERK5 substrates are nuclear transcriptional factors, whereas very few ERK5 cytosolic substrates have been characterized so far. Although ERK5 silencing affects the phosphorylation state of cytosolic proteins such as Akt and p90RSK kinases or the pro-apoptotic protein BAD, this is controversial since a direct ERK5 phosphorylation of these proteins has not been shown. For instance, Ranganathan et al. described that ERK5 phosphorylates p90RSK in vitro (Ranganathan et al., 2006), but other authors have shown that the MEK1/2 inhibitor PD184352 blocks p90RSK activation in response to EGF, at concentrations that do not block ERK5 activity (Mody et al., 2001). The use of the new synthesized specific ERK5 inhibitors—such as the XMD8-92 compound—will help to address these controversies.

ERK5 phosphorylates the transcription factor Sap1, a member of ternary complex factors (TCFs). ERK5-mediated Sap1 phosphorylation activates transcription through the Serum Response Element (SRE), which induces the expression of c-Fos (Kamakura et al., 1999). Although a direct phosphorylation by ERK5 has not been shown, co-expression of ERK5 and catalytically active MEK5 in COS-7 cells induces phosphorylation and stabilization of c-Fos and Fra-1 transcription factors (Terasawa et al., 2003). Activation of the ERK5 pathway results in phosphorylation of few Ser/Thr residues in c-Fos and Fra-1, generating more stable proteins and enhanced transactivation activity of these factors (Terasawa et al., 2003).

The three members of the MEF2 (myocyte enhancer factor-2) family of transcription factors MEF2A, MEF2C, and MEF2D are the best characterized ERK5 substrates. MEF2 proteins regulate cell differentiation in myocytes and neurons (Potthoff and Olson, 2007), and act as a nodal point for stress-response in adult tissues (Kim et al., 2008). An interaction of MEF2C with ERK5 has been shown in two hybrid and co-immunoprecipitation assays. To our knowledge, MEF2C is the only nuclear protein whose interaction with ERK5 has been mapped. MEF2C interacts with a region of the C-terminal tail of ERK5 (aa 440–501) through its N-terminal end (Yang et al., 1998; Kasler et al., 2000). More importantly, activation of ERK5 pathway by either serum or EGF stimulates the transactivation activity of the MEF2A, MEF2C, and MEF2D transcription factors. Furthermore, ERK5 seems to be determinant for EGF-stimulated activation of MEF2A and MEF2D, since expression of ERK5 dominant negative mutant abolishes this activation (Kato et al., 2000). ERK5 phosphorylates different sites in MEF2A, MEF2C, and MEF2D proteins, however many of these residues are conserved in all three members, suggesting the existence of additional structural determinants to achieve recognition of the phosphorylated sites. Thus, ERK5 phosphorylates Thr312, Thr319, and S355 in MEF2A, Ser386/7 in MEF2C, and Ser179 and in MEF2D, and mutation to alanine of these residues abrogates MEF2 transcriptional activity (Kato et al., 1997, 2000).

The promyelocytic leukemia protein (PML) is a transcription factor that acts as a tumor suppressor, inhibiting proliferation and inducing cellular senescence and apoptosis through activation of the CDK inhibitor p21 (Bernardi and Pandolfi, 2007). Yang et al. have shown that ERK5 interacts with PML at the nuclear bodies in cancer cells, and inhibits its tumor suppressor activity by phosphorylating PML protein at Ser403 and Thr409 (Yang et al., 2010). ERK5-mediated phosphorylation impairs PML-dependent activation of p21, through disrupting PML-MDM2 interaction, and downregulating expression of the p53 tumor suppressor (Yang et al., 2013).

ERK5 acts as a transcriptional co-activator, regulating MEF2C, AP-1, and c-Fos transcriptional activities in the nucleus. Therefore, translocation of ERK5 to nucleus is essential to regulate ERK5-mediated gene transcription. ERK5 is a big protein (110 kDa) so it cannot enter the nucleus by passive diffusion through the nuclear pores, as described for small proteins.

Recently, we have shown that inactive ERK5 binds the cytoplasmatic chaperone Hsp90 and the co-chaperone cell division-cycle 37 (Cdc37), which helps Hsp90 in the stabilization of ERK5 (Erazo et al., 2013). Cdc37 is the co-chaperone that specifically promotes association of Hsp90 with many protein kinases (Smith and Workman, 2009). This trimeric complex, ERK5-Hsp90-Cdc37, not only stabilizes inactive ERK5, but also keeps ERK5 in a suitable conformation for MEK5 recognition and activation (Erazo et al., 2013).

In basal conditions, ERK5 binds the cytoplasmatic chaperone Hsp90, which serves as a cytosolic anchor for ERK5 (Erazo et al., 2013). This inactive ERK5 adopts a closed conformation where the C-terminal tail interacts with the kinase domain and the NLS motif is hidden and not available for the nuclear transport (reviewed in Kondoh et al., 2006). Nuclear shuttling of ERK5 requires both a conformational change to allow exposure of the NLS motif and the release of Hsp90. This mechanism is analogous to the one described for the progesterone and androgen receptors, which also requires Hsp90 dissociation for their nuclear translocation (Picard, 2006).

So far, two different mechanisms have been proposed for the ERK5 shuttling to the nucleus: one of them requires C-terminal phosphorylation, while the other does not. Once in the nucleus, ERK5 enhances gene transcription by either phosphorylating transcription factors, or by interacting with these factors through the transactivation TAD domain located at the C-terminal. Strikingly, ERK5 does not require kinase activity to interact with and activate transcription factors and therefore, forms of nuclear ERK5 devoid of kinase activity are able to activate transcription (Diaz-Rodriguez and Pandiella, 2010; Inesta-Vaquera et al., 2010; Erazo et al., 2013). Kinase-independent nuclear functions have been also proposed for other MAP kinases that lack a TAD domain. For instance, ERK2 promotes cell cycle entry by disrupting retinoblastoma-lamin A complexes in a kinase-independent fashion (Rodriguez et al., 2010), and also binds DNA acting as a transcriptional repressor for interferon gamma-induced genes (Hu et al., 2009). It would be interesting to study if ERK5 can also bind DNA.

During the last years, efforts of many laboratories have led to delineate the importance of ERK5 in controlling cell proliferation in normal and cancer cells, by mechanisms that are both dependent and independent of its kinase activity: nuclear ERK5 activates transcription factors by either direct phosphorylation or acting as co-activator thanks to a unique transcriptional activation domain located at its C-terminal tail. Consequently, ERK5 has been proposed as an interesting target to tackle different cancers, and either inhibitors of ERK5 activity or silencing the protein have shown antiproliferative activity in cancer cells and to block tumor growth in animal models. However, and as we have seen above, ERK5 kinase inhibitors such as XMD8-92 might not be useful in cancers showing kinase-inactive nuclear ERK5. On the other hand, the anticancer activity of ERK5 inhibitors should be carefully interpreted. It has been recently reported that several classes of kinase inhibitors can strongly inhibit the bromodomain-containing protein-4 Brd4, a general transcription co-activator (Ciceri et al., 2014). This is the case for XMD8-92, which inhibits Brd4 and ERK5 with similar potency (Lin et al., 2016), and therefore some of the antitumor effects of this compound could be mediated by the Brd4-inhibiting activity. A new generation of specific ERK5 inhibitors are required to clarify the exact role of ERK5 in cancer cell growth.

There are still many open questions that remain to be addressed in order to describe the precise mechanism involved in ERK5 nuclear shuttling. For instance, is CDK1 the only kinase that phosphorylates ERK5 C-terminal tail? Does Cdc37 overexpression mimic a physiological mechanism that allows nuclear shuttling of inactive ERK5? Are other post-translational modifications, such as SUMOylation, required for ERK5 nuclear translocation? What is the role of ERK5 phosphatases in this process? The very near future seems an exciting time to deal with these questions, and hopefully will provide clues to design new compounds with antiproliferative activity.

All three authors wrote and corrected the manuscript. TE desgined Table 1. JL designed Figure 1.