Characterization of the reversible phosphorylation and activation of ERK8

Iva V. Klevernic, Margaret J. Stafford, Nicholas Morrice, Mark Peggie, Simon Morton, Philip Cohen

    Research output: Contribution to journalArticle

    41 Citations (Scopus)

    Abstract

    ERK8 (extracellular-signal-regulated protein kinase 8) expressed in Escherichia coli or insect cells was catalytically active and phosphorylated at both residues of the Thr-Glu-Tyr motif. Dephosphorylation of the threonine residue by PP2A (protein serine/threonine phosphatase 2A) decreased ERK8 activity by over 95 % in vitro, whereas complete dephosphorylation of the tyrosine residue by PTP1B (protein tyrosine phosphatase 1B) decreased activity by only 15-20%. Wild-type ERK8 expressed in HEK-293 cells was over 100-fold less active than the enzyme expressed in bacteria or insect cells, but activity could be increased by exposure to hydrogen peroxide, by incubation with the protein serine/threonine phosphatase inhibitor okadaic acid, or more weakly by osmotic shock. In unstimulated cells, ERK8 was monophosphorylated at Tyr-177, and exposure to hydrogen peroxide induced the appearance of ERK8 that was dually phosphorylated at both Thr-175 and Tyr-177. IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor), PMA or anisomycin had little effect on activity. In HEK-293 cells, phosphorylation of the Thr-Glu-Tyr motif of ERK8 was prevented by Ro 318220, a potent inhibitor of ERK8 in vitro. The catalytically inactive mutants ERK8[D154A] and ERK8[K42A] were not phosphorylated in HEK-293 cells or E. coli, whether or not the cells had been incubated with protein phosphatase inhibitors or exposed to hydrogen peroxide. Our results suggest that the activity of ERK8 in transfected HEK-293 cells depends on the relative rates of ERK8 autophosphorylation and dephosphorylation by one or more members of the PPP family of protein serine/threonine phosphatases. The major residue in myelin basic protein phosphorylated by ERK8 (Ser-126) was distinct from that phosphorylated by ERK2 (Thr-97), demonstrating that, although ERK8 is a proline-directed protein kinase, its specificity is distinct from ERK1/ERK2.

    Original languageEnglish
    Pages (from-to)365-373
    Number of pages9
    JournalBiochemical Journal
    Volume394
    Issue number1
    DOIs
    Publication statusPublished - 15 Feb 2006

    Fingerprint

    Phosphorylation
    Protein Kinases
    Chemical activation
    HEK293 Cells
    Phosphoprotein Phosphatases
    Hydrogen Peroxide
    human ERK8 protein
    Escherichia coli
    Insects
    Proline-Directed Protein Kinases
    Non-Receptor Type 1 Protein Tyrosine Phosphatase
    Anisomycin
    Okadaic Acid
    Myelin Basic Protein
    Osmotic Pressure
    Somatomedins
    Threonine
    Epidermal Growth Factor
    Tyrosine
    Bacteria

    Keywords

    • Extracellular-signal-regulated kinase 8 (ERK8)
    • Mass spectrometry
    • Mitogen-activated protein kinase (MAPK)
    • Oxidative stress
    • Protein phosphatase

    Cite this

    Klevernic, I. V., Stafford, M. J., Morrice, N., Peggie, M., Morton, S., & Cohen, P. (2006). Characterization of the reversible phosphorylation and activation of ERK8. Biochemical Journal, 394(1), 365-373. https://doi.org/10.1042/BJ20051288
    Klevernic, Iva V. ; Stafford, Margaret J. ; Morrice, Nicholas ; Peggie, Mark ; Morton, Simon ; Cohen, Philip. / Characterization of the reversible phosphorylation and activation of ERK8. In: Biochemical Journal. 2006 ; Vol. 394, No. 1. pp. 365-373.
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    abstract = "ERK8 (extracellular-signal-regulated protein kinase 8) expressed in Escherichia coli or insect cells was catalytically active and phosphorylated at both residues of the Thr-Glu-Tyr motif. Dephosphorylation of the threonine residue by PP2A (protein serine/threonine phosphatase 2A) decreased ERK8 activity by over 95 {\%} in vitro, whereas complete dephosphorylation of the tyrosine residue by PTP1B (protein tyrosine phosphatase 1B) decreased activity by only 15-20{\%}. Wild-type ERK8 expressed in HEK-293 cells was over 100-fold less active than the enzyme expressed in bacteria or insect cells, but activity could be increased by exposure to hydrogen peroxide, by incubation with the protein serine/threonine phosphatase inhibitor okadaic acid, or more weakly by osmotic shock. In unstimulated cells, ERK8 was monophosphorylated at Tyr-177, and exposure to hydrogen peroxide induced the appearance of ERK8 that was dually phosphorylated at both Thr-175 and Tyr-177. IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor), PMA or anisomycin had little effect on activity. In HEK-293 cells, phosphorylation of the Thr-Glu-Tyr motif of ERK8 was prevented by Ro 318220, a potent inhibitor of ERK8 in vitro. The catalytically inactive mutants ERK8[D154A] and ERK8[K42A] were not phosphorylated in HEK-293 cells or E. coli, whether or not the cells had been incubated with protein phosphatase inhibitors or exposed to hydrogen peroxide. Our results suggest that the activity of ERK8 in transfected HEK-293 cells depends on the relative rates of ERK8 autophosphorylation and dephosphorylation by one or more members of the PPP family of protein serine/threonine phosphatases. The major residue in myelin basic protein phosphorylated by ERK8 (Ser-126) was distinct from that phosphorylated by ERK2 (Thr-97), demonstrating that, although ERK8 is a proline-directed protein kinase, its specificity is distinct from ERK1/ERK2.",
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    Klevernic, IV, Stafford, MJ, Morrice, N, Peggie, M, Morton, S & Cohen, P 2006, 'Characterization of the reversible phosphorylation and activation of ERK8', Biochemical Journal, vol. 394, no. 1, pp. 365-373. https://doi.org/10.1042/BJ20051288

    Characterization of the reversible phosphorylation and activation of ERK8. / Klevernic, Iva V.; Stafford, Margaret J.; Morrice, Nicholas; Peggie, Mark; Morton, Simon; Cohen, Philip.

    In: Biochemical Journal, Vol. 394, No. 1, 15.02.2006, p. 365-373.

    Research output: Contribution to journalArticle

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    Klevernic IV, Stafford MJ, Morrice N, Peggie M, Morton S, Cohen P. Characterization of the reversible phosphorylation and activation of ERK8. Biochemical Journal. 2006 Feb 15;394(1):365-373. https://doi.org/10.1042/BJ20051288