Erk3 and Erk4
Extracellular signal-regulated kinase 3 (ERK3) and ERK4 are atypical members of the mitogen-activated protein (MAP) kinase family of serine/threonine kinases. The ERK3 and ERK4 genes were originally identified in 1991 and 1992, by homology cloning with probes derived from the MAP kinase ERK1 (Gonzalez et al. 2002; Boulton et al. 1991). In human, ERK3 is encoded by the MAPK6 gene located on chromosome 15q21.2. The MAPK4 gene present on chromosome 18q21.1 encodes ERK4. The high sequence identity of ERK3 and ERK4 proteins and the similar organization of their genes indicate that the two proteins are true paralogs.
Structure of ERK3 and ERK4
Regulation of ERK3 and ERK4 Expression and Localization
ERK3 is expressed ubiquitously in adult mammalian tissues, whereas ERK4 shows a more restricted expression profile. The level of expression of the two kinases varies considerably between tissues, but the highest expression is found in the brain. In the mouse embryo, they share a similar temporal pattern of regulation with a peak of expression at embryonic day 11, coincident with the time of early organogenesis (Rousseau et al. 2010). During thymocyte differentiation, transcription of the ERK3 gene is apparent at DN1 stage and increases up to DN4 stage, followed by a decrease at the DP stage and in SP thymocytes (Marquis et al. 2014).
Interestingly, ERK3 and ERK4 proteins display different stability. Whereas ERK4 is a relatively stable protein, ERK3 is highly unstable with a half-life of 30–60 min in proliferating cells (Kant et al. 2006; Aberg et al. 2006; Coulombe et al. 2003). ERK3 is constitutively degraded by the ubiquitin-proteasome system, and two regions in the N-terminal lobe of the kinase domain were found to be necessary and sufficient to target ERK3 for proteolysis (Fig. 2) (Coulombe et al. 2003). This suggests that ERK3 biological activity might be regulated by its cellular abundance. The short half-life of ERK3 has physiological significance since the protein is stabilized and accumulates to high levels during myogenic differentiation and in mitosis (Coulombe et al. 2003; Tanguay et al. 2010). The upregulation of ERK3 during skeletal muscle differentiation is concomitant to accumulation of the cell cycle inhibitor p21Cip1 and cell cycle exit. In mitosis, the phosphorylation of ERK3 on four residues in the C-terminal extension by cyclin B-CDK1 stabilizes the protein and leads to its transient accumulation (Tanguay et al. 2010). Another study reported that ERK3 is hydroxylated on proline 25 by prolyl hydroxylase 3, a modification that protects ERK3 from proteasomal degradation (Rodriguez et al. 2016).
At the subcellular level, ERK3 and ERK4 proteins exhibit distinct localization. ERK3 is found in both the cytoplasm and the nucleus of a variety of cell types, whereas ERK4 appear to localize mainly in the cytoplasmic compartment. The cytoplasmic localization of ERK3 and ERK4 requires an active nuclear export by a CRM1-dependent mechanism. In contrast to classical MAP kinases, the cellular distribution of ERK3 and ERK4 does not change in response to common mitogenic or stress stimuli. However, ERK3 localization is regulated through its interaction with MAP kinase-activated protein kinase 5 (MK5) (Seternes et al. 2004; Schumacher et al. 2004). MK5 is a member of the MAP kinase-activated protein kinase (MK) family of protein kinases that lie downstream of MAP kinase signaling pathways. The formation of a complex between ERK3 and MK5 results in the nuclear to cytoplasmic redistribution of both proteins. The kinase activity of either protein is dispensable for this relocalization. In addition to its effect on localization, MK5 also regulates ERK3 expression. Mouse embryonic fibroblasts prepared from MK5-deficient mice or HeLa cells depleted of MK5 by RNA interference show a marked reduction of ERK3 protein levels (Seternes et al. 2004).
Regulation of ERK3 and ERK4 Kinase Activity
The regulation of ERK3 and ERK4 kinase activity remains superficially understood. ERK3 and ERK4 are phosphorylated on Ser189 and Ser186, respectively, in the Ser-Glu-Gly motif of their activation loop (Aberg et al. 2006; Coulombe et al. 2003). Unlike classical MAP kinases, this phosphorylation event is detected in resting cells and is not modulated by common mitogenic or stress stimuli. Group I p21-activated kinases (PAK1, PAK2, and PAK3) were identified as activation loop kinases of ERK3 and ERK4 (Déléris et al. 2011). PAK-mediated phosphorylation of ERK3 and ERK4 results in their enzymatic activation (Fig. 2). Specific stimuli that modulate the kinase activity of ERK3 or ERK4 are yet to be identified.
Substrates and Physiological Functions of ERK3 and ERK4
Much remains to be learned about the physiological functions of ERK3 and ERK4. However, in vivo analysis of mice deficient for ERK3 or ERK4 and in vitro studies in different cell culture models have started to shed light on their potential roles. These studies suggest that ERK3 and ERK4 are involved in the control of cell differentiation, cytoskeletal remodeling, cell migration and invasion, DNA repair, and immune response (Fig. 3). Genetic disruption of Mapk6 in mice leads to intrauterine growth restriction, delayed lung maturation associated with decreased sacculation and defective type II pneumocyte differentiation, and neonatal lethality (Klinger et al. 2009). Whereas the lung maturation defect can be overcome by in utero glucocorticoid administration, the newborn mice cannot be rescued from neonatal death. This indicates that additional physiological alterations contribute to the neonatal lethality. ERK3-deficient mice have reduced levels of insulin-like growth factor 2 (IGF-2) in the serum, suggesting that ERK3 might regulate IGF-2 levels. ERK3 signaling also plays a role in thymocyte development and T cell activation. Analysis of the thymus of newborn mice revealed that loss of ERK3 is associated with a 50% decrease in the number of CD4+ CD8+ (DP) thymocytes. ERK3 deficiency results in a decrease in DP thymocytes’ half-life and an impairment in the ability of these cells to make successful T-cell receptor α gene rearrangements (Marquis et al. 2014). Reconstitution of the lymphoid compartment with ERK3-deficient fetal liver cells occurs normally in hematopoietic chimeras. ERK4-deficient mice are viable, develop normally, and show no gross physiological anomalies (Rousseau et al. 2010). Interestingly, behavioral analysis revealed that ERK4 mutant mice manifest depression-like behavior. In these mice, the loss of ERK4 is not compensated by increased activity or level of ERK3.
Results from in vitro studies have shown that ERK3 expression is upregulated during differentiation of P19 and PC-12 cells into neurons, and during myogenic differentiation of C2C12 myoblasts, concomitant to cell cycle exit (Coulombe et al. 2003; Boulton et al. 1991). A possible role of ERK3 in cell cycle regulation is suggested by a number of observations. ERK3 was found to interact with the cell cycle regulatory proteins cyclin D3, cell-division cycle 14A (CDC14A), and CDC14B through its C-terminal extension (Sun et al. 2006; Hansen et al. 2008; Tanguay et al. 2010). Also, the level and phosphorylation state of ERK3 vary during cell cycle progression. ERK3 is phosphorylated in the C-terminal extension during entry into mitosis and dephosphorylated at the M- to G1-phase transition. CDC14A and CDC14B phosphatases reverse the C-terminal phosphorylation of ERK3, whereas the mitotic kinase cyclin B1-CDK1 is most likely responsible for the phosphorylation of these sites (Tanguay et al. 2010).
Accumulating evidence suggests that ERK3/ERK4 signaling controls actin cytoskeleton dynamics, cellular morphogenesis, and cell migration. In primary hippocampal neurons, ERK3 forms a ternary complex with MK5 and the cytoskeletal protein septin 7, which may facilitate the phosphorylation of the septin-interacting proteins Binder of Rho GTPases (BORG) 1-3 by ERK3 and/or MK5 (Brand et al. 2012). Functionally, coexpression of ERK3, MK5, and septin 7 increases neurons branching and spine number. These results suggest a role of ERK3-MK5 signaling in neuronal morphogenesis. It has been proposed that inhibition of ERK4 translation by IGF2BP1 attenuates MK5 activation and prevents phosphorylation of HSP27, resulting in decreased sequestration of actin monomers and, consequently, promotion of cell migration (Stöhr et al. 2012). ERK3 was also reported to interact with and phosphorylate steroid receptor coactivator 3 (SRC-3), resulting in upregulation of MMP gene expression and proinvasive activity in lung cancer cells (Long et al. 2012). Silencing of ERK3 expression decreases the ability of lung cancer cells to metastasize to the lungs in the mouse tail vein injection model, suggesting a possible role of ERK3 in tumor progression. Another study reported that ERK3 promotes endothelial cell migration, proliferation, and tube formation by inducing SRC-3-dependent vascular endothelial growth factor (VEGF) receptor 2 expression (Wang et al. 2014).
ERK3 and ERK4 were among the first MAP kinases to be identified in the early 1990s. However, the characterization of these protein kinases has lagged behind that of classical MAP kinase family members such as ERK1/ERK2. Much still remains to be learned about their regulation, the identity and spectrum of their substrates, their physiological roles, and their putative involvement in human diseases. The identification of small molecule inhibitors of ERK3 and ERK4 will be essential to address these questions and should be a priority for the field.
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