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PLoS One. 2015 Feb 17;10(2):e0117914. doi: 10.1371/journal.pone.0117914. eCollection 2015.

Phosphorylation of ERK5 on Thr732 is associated with ERK5 nuclear localization and ERK5-dependent transcription.

Author information

  • 1Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan.
  • 2Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan; Department of Pharmacology, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata, 990-9585, Japan.
  • 3Cell Signaling Technology, 3 Trask Lane, Danvers, MA 01923, United States of America.
  • 4Department of Pharmacology, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata, 990-9585, Japan.

Abstract

Extracellular signal-regulated kinases (ERKs) play critical roles in numerous cellular processes, including proliferation and differentiation. ERK5 contains a kinase domain at the N-terminal, and the unique extended C-terminal includes multiple autophosphorylation sites that enhance ERK5-dependent transcription. However, the impact of phosphorylation at the various sites remain unclear. In this study, we examined the role of phosphorylation at the ERK5 C-terminal. We found that a constitutively active MEK5 mutant phosphorylated ERK5 at the TEY motif, resulting in the sequential autophosphorylation of multiple C-terminal residues, including Thr732 and Ser769/773/775. However, when ERK1/2 was selectively activated by an oncogenic RAS mutant, ERK5 phosphorylation at Thr732 was induced without affecting the phosphorylation status at TEY or Ser769/773/775. The Thr732 phosphorylation was U0126-sensitive and was observed in a kinase-dead mutant of ERK5 as well, suggesting that ERK1/2 can phosphorylate ERK5 at Thr732. This phosphorylation was also promoted by epidermal growth factor and nerve growth factor in HEK293 and PC12 cells, respectively. The ERK5-T732A mutant was localized in the cytosol under basal conditions. In contrast, ERK5 phosphorylated at Thr732 via the RAS-ERK1/2 pathway and ERK5-T732E, which mimics the phosphorylated form, were localized in both the nucleus and cytosol. Finally, ER-32A and U0126 blocked ERK5-dependent MEF2C transcriptional activity. Based on these findings, we propose a novel cross-talk mechanism in which ERK1/2, following activation by growth factor stimulation, phosphorylates ERK5 at Thr732. This phosphorylation event is responsible for ERK5 nuclear localization and ERK5-dependent transcription.

PMID:
25689862
PMCID:
PMC4331489
DOI:
10.1371/journal.pone.0117914
[PubMed - indexed for MEDLINE]
Free PMC Article
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