Activation of a C-terminal Transcriptional Activation Domain of ERK5 by Autophosphorylation

Abstract

ERK5 plays a crucial role in many biological processes by regulating transcription. ERK5 has a large C-terminal-half that contains a transcriptional activation domain. However, it has remained unclear how its transcriptional activation activity is regulated. Here, we show that the activated kinase activity of ERK5 is required for the C-terminal-half to enhance the AP-1 activity, and that the activated ERK5 undergoes autophosphorylation on its most C-terminal region. Changing these phosphorylatable threonine and serine residues to unphosphorylatable alanines significantly reduces the transcriptional activation activity of ERK5. Moreover, phosphomimetic mutants of the C-terminal-half of ERK5 without an N-terminal kinase domain are shown to be able to enhance the AP-1 activity in fibroblastic cells. These results reveal the role of the stimulus-induced ERK5 autophosphorylation in regulation of gene expression. The mitogen-activated protein kinase (MAPK)2 cascades play an essential role in transducing extracellular signals to cytoplasmic and nuclear effectors, and regulate a variety of cellular functions, including cell proliferation, differentiation, and stress responses. The MAPK cascades are composed of three classes of protein kinases: MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Each MAPK is activated by specific members of MAPKK, which are activated by MAPKKK (1-13). Four subfamily members of the MAPK family have been relatively well studied; extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38, and ERK5.ERK5 is activated by oxidative stress, hyperosmolarity, and several growth factors including epidermal growth factor and nerve growth factor (14 -18). MEK5 is the upstream MAPKK that specifically phosphorylates and activates ERK5 (16,19). It has been shown that ERK5 directly phosphorylates and activates several transcription factors including c-Myc, Sap1a, c-Fos, Fra-1, and MEF2 family members (15, 17, 20 -22). ERK5 knock-out mice die in embryonic stages with angiogenic failure and cardiovascular defects, and in the adult stages, ERK5 is required for endothelial cell survival and maintenance of blood vascular integrity (23-26). The targeted deletion of MEK5 also causes early embryonic death with cardiovascular defects (27). Moreover, knockdown studies in Xenopus have shown that ERK5 and MEK5 play an essential role in neural differentiation (28).ERK5 has a large, unique C-terminal-half not found in other MAPK family members (19,29). Because of this unique 400-amino acid extension, ERK5 is also called big MAP kinase 1 (BMK1). The C-terminal-half of ERK5 has two proline-rich domains and a bipartite NLS, and shares no high homology with other proteins (19,30,31). Kasler et al. (32) demonstrated that the C-terminal-half of ERK5 has a potent transcriptional activation domain. It was subsequently shown that the ERK5 C-terminal-half is essential for transactivation of MEF2C (30). Moreover, a recent study has shown that ERK5 has two transactivation domains in its C-termin...

Multisite phosphorylation of Erk5 in mitosis

“…MEK5 is responsible for the phosphorylation of Erk5 at the TEY microdomain (Zhou et al, 3148 Journal of Cell Science 123 (18) 1995; Nishimoto and Nishida, 2006). In addition, a report indicated that once Erk5 in phosphorylated at the T-PEY-P sites, it is then able to autophosphorylate at residues located in its C-terminal region (Morimoto et al, 2007). We explored the participation of both kinases as the potential Erk5 mitotic kinase.…”

“…Within this region of Erk5, Morimoto and collaborators (Morimoto et al, 2007) had identified several residues phosphorylated by active Erk5 after transfection of a constitutively activated MEK5 form (MEK5DD). Even though our data excluded MEK5 or Erk5 as the C-terminal mitotic kinases, the possibility that those residues were the targets for such a phosphorylation, due perhaps to a predisposition for being phosphorylated, led us to create specific point-mutants in which those potentially phosphorylated residues were mutated to non-phosphorylatable ones (alanine residues).…”

“…In addition, this region contains a potent transcriptional activation domain (Kasler et al, 2000). A recent report indicated that Erk5 undergoes autophosphorylation on this C-terminal region and this process could be important in regulating gene expression, since it would enhance the AP-1 activity (Morimoto et al, 2007). In addition, this C-terminal zone has been implicated in the nucleocytoplasmic trafficking of Erk5 (Borges et al, 2007).…”

MAPK7 (mitogen-activated protein kinase 7)

“…In cardiac tissue, ERK5 may couple cells electrically and metabolically by phosphorylating the gap-junction protein Cx43 at a key residue for gap junction communication (Cameron et al, 2003). Also, phosphorylated ERK5 regulates gene expression through its C-terminal transcriptional activation domain (Morimoto et al, 2007).…”

“…The activation of ERK5 occurs via interaction with and dual phosphorylation in its TEY motif by MKK5 (Mody et al, 2003). MKK5 mediated ERK5 activation leads to ERK5 autophosphorylation in its unique C-terminal domain (Morimoto et al, 2007).…”