Characterization of the Atypical MAPK ERK4 and Its Activation of the MAPK-activated Protein Kinase MK5

Abstract

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...

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