Extracellular-regulated kinase 3 (ERK3, MAPK6) is an atypical member of the ERKs, lacking the threonine and tyrosine residues in the activation loop, carrying a unique C-terminal extension and being mainly regulated by its own protein stability and/or by autophosphorylation. Here we show that ERK3 specifically interacts with the MAPKactivated protein kinase 5 (MK5 or PRAK) in vitro and in vivo. Expression of ERK3 in mammalian cells leads to nuclear-cytoplasmic translocation and activation of MK5 and to phosphorylation of both ERK3 and MK5. Remarkably, activation of MK5 is independent of ERK3 enzymatic activity, but depends on its own catalytic activity as well as on a region in the C-terminal extension of ERK3. In mouse embryonic development, mRNA expression patterns of ERK3 and MK5 suggest spatiotemporal coexpression of both kinases. Deletion of MK5 leads to strong reduction of ERK3 protein levels and embryonic lethality at about stage E11, where ERK3 expression in wild-type mice is maximum, indicating a role of this signalling module in development.

The extracellular-regulated kinase (ERK) 4 (MAPK4) and ERK3 (MAPK6) are structurally related atypical MAPKs displaying major differences only in the C-terminal extension. ERK3 is known as an unstable mostly cytoplasmic protein that binds, translocates, and activates the MAPK-activated protein kinase (MK) 5. Here we have investigated the stability and expression of ERK4 and have analyzed its ability to bind, translocate, and activate MK5. We show that, in contrast to ERK3, ERK4 is a stable protein that binds to endogenous MK5. Interaction of ERK4 with MK5 leads to translocation of MK5 to the cytoplasm and to its activation by phosphorylation. In transfected HEK293 cells, where overexpressed catalytically dead ERK3 is able to activate MK5, catalytic activity of ERK4 is necessary for activation of MK5, indicating that ERK4 directly phosphorylates MK5. Interestingly, ERK4 dimerizes and/or oligomerizes with ERK3, suggesting that overexpressed inactive ERK3 recruits active endogenous ERK4 to MK5 for its activation. Hence, ERK3 and ERK4 cooperate in activation of MK5. Mitogen-activated protein kinases (MAPKs)2 represent a family of evolutionary conserved enzymes with a central role in the well characterized MAPK signaling cascades. A wide variety of extracellular stimuli serve as activators of MAPK pathways leading to appropriate responses of cells, such as proliferation, differentiation, growth, and migration. MAPK pathways generally have a three-kinase module architecture by which the signal is transmitted from an upstream kinase to a downstream kinase by sequential phosphorylation. MAPKs comprise four well defined groups (ERK1/2 (1, 2), c-Jun N-terminal kinases, p38s, and ERK5 (BMK) (3)), but additional members including ERK3 (1, 4), ERK4 (p63 MAPK, ERK3-related, ERK3␤, MAPK4, Prkm4) (5), and ERK8 (6) have been identified. ERK4 (p63 MAPK) was described in humans (5), soon after ERK1, ERK2, and ERK3 were identified (1). Among MAPKs, ERK4 is most closely related to ERK3 displaying 62% overall amino acid sequence identity and 73% within the predicted kinase domain. Both kinases do not contain the highly conserved activation loop ("a-loop") motif TXY between kinase subdomains VII and VIII that is found in all other MAPKs but possess a SEG sequence instead (Fig. 1A). Even the APE motif of subdomain VIII, which is extremely conserved in other MAPKs, is replaced by an SPR motif in ERK3 and ERK4 (Fig. 1A). ERK4 and ERK3 carry long C-terminal extensions (Fig. 1B). Human Erk4 was mapped on chromosome 18q12-21 (7), and a cDNA for the rat homolog rMNK2 was isolated (8). Stimuli, activators, or relevant substrates of ERK4 have remained elusive, and enzymatic activities of the atypical ERKs have not been well defined so far.Initially the MAPK-activated protein kinase MK5 (9, 10), also known as p38-regulated and -activated kinase (PRAK), was described as a member of the MK family and a downstream target of p38 (for recent reviews see Refs. 11 and 12). Previous data suggested that MK5 is not a physiological substrate for p38 in viv...

Mitogen-activated protein kinase-activated protein (MAPKAP) kinase 5 (MK5) deficiency is associated with reduced extracellular signal-regulated kinase 3 (ERK3) (mitogen-activated protein kinase 6) levels, hence we utilized the MK5 knockout mouse model to analyze the physiological functions of the ERK3/MK5 signaling module. MK5-deficient mice displayed impaired dendritic spine formation in mouse hippocampal neurons in vivo. We performed large-scale interaction screens to understand the neuronal functions of the ERK3/MK5 pathway and identified septin7 (Sept7) as a novel interacting partner of ERK3. ERK3/MK5/ Sept7 form a ternary complex, which can phosphorylate the Sept7 regulators Binders of Rho GTPases (Borgs). In addition, the brain-specific nucleotide exchange factor kalirin-7 (Kal7) was identified as an MK5 interaction partner and substrate protein. In transfected primary neurons, Sept7-dependent dendrite development and spine formation are stimulated by the ERK3/MK5 module. Thus, the regulation of neuronal morphogenesis is proposed as the first physiological function of the ERK3/MK5 signaling module. E xtracellular signal-regulated kinase 3 (ERK3) (mitogen-activated protein kinase 6 [MAPK6]) and ERK4 (MAPK4) belong to the group of atypical MAPKs which display a SEG motif in the activation loop (instead of TEY) and carry a long C-terminal extension (1, 15, 58). The regulation, substrate specificity, and physiological functions of atypical MAP kinases are not completely understood (7). The phosphorylation of ERK3 and ERK4 at the serine residue in their activation loop proceeds through upstream protein kinase(s), such as the recently identified p21-activated protein kinases (PAKs) (8, 10), and leads to their activation (6, 9, 37). ERK3 also interacts with the protein phosphatase Cdc14A and is probably an in vivo substrate for this enzyme (16). The recent targeted deletion of ERK3 in mouse indicates that this enzyme is essential for neonatal survival and critical for the establishment of fetal growth potential and pulmonary function. The surviving ERK3-deficient pups show reduced reflexes and diminished ability to suckle (25). In contrast, the targeted inactivation of ERK4 in mice does not compromise the embryonic development, viability, and fertility of these animals and does not exacerbate the ERK3 phenotype, but it leads to a depression-related behavior in a forced swimming test (38).Only one substrate has been described for ERK3 and ERK4 so far, namely, the MAPK-activated protein (MAPKAP) kinase MK5 (also known as PRAK) (1,19,41,42). MK5 binds to ERK3 and ERK4 via a novel MAPK interaction motif (2). An increased level of cytoplasmic ERK3 causes the nuclear-cytoplasmic translocation of MK5, the formation of ERK3/MK5 signaling complexes, and the subsequent activation of MK5 by phosphorylation. The findings that the small interfering RNA (siRNA)-mediated knockdown of ERK3 reduces intracellular MK5 activity (1) and that the ERK3 level is reduced in MK5-deficient cells (41) clearly demonstrate the functional exis...

The structurally related MAPK-activated protein kinases (MAPKAPKs or MKs) MK2, MK3 and MK5 are involved in multiple cellular functions, including cell-cycle control and cellular differentiation. Here, we show that after deregulation of cell-cycle progression, haematopoietic stem cells (HSCs) in MK2-deficient mice are reduced in number and show an impaired ability for competitive repopulation in vivo. To understand the underlying molecular mechanism, we dissected the role of MK2 in association with the polycomb group complex (PcG) and generated a MK2 mutant, which is no longer able to bind to PcG. The reduced ability for repopulation is rescued by re-introduction of MK2, but not by the Edr2-nonbinding mutant of MK2. Thus, MK2 emerges as a regulator of HSC homeostasis, which could act through chromatin remodelling by the PcG complex.

Synaptic inhibition in the spinal cord is mediated mainly by strychnine-sensitive glycine (GlyRs) and by γ-aminobutyric acid type A receptors (GABAAR). During neuronal maturation, neonatal GlyRs containing α2 subunits are replaced by adult-type GlyRs harboring α1 and α3 subunits. At the same time period of postnatal development, the transmembrane chloride gradient is changed due to increased expression of the potassium-chloride cotransporter (KCC2), thereby shifting the GABA- and glycine-mediated synaptic currents from mostly excitatory depolarization to inhibitory hyperpolarization. Here, we used RNA interference to suppress KCC2 expression during in vitro maturation of spinal cord neurons. Morphological analysis revealed reduced numbers and size of dendritic GlyR clusters containing α1 subunits but not of clusters harboring neonatal α2 subunits. The morphological changes were accompanied by decreased frequencies and amplitudes of glycinergic miniature inhibitory currents, whereas GABAergic synapses appeared functionally unaltered. Our data indicate that KCC2 exerts specific functions for the maturation of glycinergic synapses in cultured spinal cord neurons.