Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2

R. Ben-Levy, I. A. Leighton, Y. N. Doza, P. Attwood, N. Morrice, C. J. Marshall, P. Cohen

    Research output: Contribution to journalArticle

    152 Citations (Scopus)

    Abstract

    MAP kinase-activated protein (MAPKAP) kinase-2 is activated in vivo by reactivating kinase (RK), a MAP kinase (MAPK) homologue stimulated by cytokines and cellular stresses. Here we show that in vitro RK phosphorylates human GST-MAPKAP kinase-2 at Thr25 in the proline-rich N-terminal region, Thr222 and Ser272 in the catalytic domain and Thr334 in the C-terminal domain. Using novel methodology we demonstrate that activation of MAPKAP kinase-2 requires the phosphorylation of any two of the three residues Thr222, Ser272 and Thr334. Ser9, Thr25, Thr222, Ser272, Thr334 and Thr338 became 32P-labelled in stressed KB cells and labelling was prevented by the specific RK inhibitor SE 203580, establishing that RK phosphorylates Thr25, Thr222, Ser272 and Thr334 in vivo. The 32P-labelling of Thr338 is likely to result from autophosphorylation. GST-MAPKAP kinase-2 lacking the N-terminal domain was inactive, but activated fully when phosphorylated at Thr222, Ser272 and Thr334 by p42 MAPK or RK. In contrast, full-length GST-MAPKAP kinase-2 was phosphorylated at Thr25 (and not activated) by p42 MAPK, suggesting a role for the N-terminal domain in specifying activation by RK in vivo. The mutant Asp222/Asp334 was 20% as active as phosphorylated MAPKAP kinase-2, and this constitutively active form may be useful for studying its physiological roles.

    Original languageEnglish
    Pages (from-to)5920-5930
    Number of pages11
    JournalEMBO Journal
    Volume14
    Issue number23
    Publication statusPublished - 1 Jan 1995

    Fingerprint

    Phosphorylation
    Phosphotransferases
    Chemical activation
    Mitogen-Activated Protein Kinase 1
    Labeling
    KB Cells
    Mitogen-Activated Protein Kinase Kinases
    Proline
    MAP-kinase-activated kinase 2
    Catalytic Domain
    Cytokines

    Keywords

    • Cytokine
    • Heat shock
    • MAP kinase
    • MAPKAP kinase
    • Stress

    Cite this

    Ben-Levy, R., Leighton, I. A., Doza, Y. N., Attwood, P., Morrice, N., Marshall, C. J., & Cohen, P. (1995). Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2. EMBO Journal, 14(23), 5920-5930.
    Ben-Levy, R. ; Leighton, I. A. ; Doza, Y. N. ; Attwood, P. ; Morrice, N. ; Marshall, C. J. ; Cohen, P. / Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2. In: EMBO Journal. 1995 ; Vol. 14, No. 23. pp. 5920-5930.
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    Ben-Levy, R, Leighton, IA, Doza, YN, Attwood, P, Morrice, N, Marshall, CJ & Cohen, P 1995, 'Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2', EMBO Journal, vol. 14, no. 23, pp. 5920-5930.

    Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2. / Ben-Levy, R.; Leighton, I. A.; Doza, Y. N.; Attwood, P.; Morrice, N.; Marshall, C. J.; Cohen, P.

    In: EMBO Journal, Vol. 14, No. 23, 01.01.1995, p. 5920-5930.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2

    AU - Ben-Levy, R.

    AU - Leighton, I. A.

    AU - Doza, Y. N.

    AU - Attwood, P.

    AU - Morrice, N.

    AU - Marshall, C. J.

    AU - Cohen, P.

    PY - 1995/1/1

    Y1 - 1995/1/1

    N2 - MAP kinase-activated protein (MAPKAP) kinase-2 is activated in vivo by reactivating kinase (RK), a MAP kinase (MAPK) homologue stimulated by cytokines and cellular stresses. Here we show that in vitro RK phosphorylates human GST-MAPKAP kinase-2 at Thr25 in the proline-rich N-terminal region, Thr222 and Ser272 in the catalytic domain and Thr334 in the C-terminal domain. Using novel methodology we demonstrate that activation of MAPKAP kinase-2 requires the phosphorylation of any two of the three residues Thr222, Ser272 and Thr334. Ser9, Thr25, Thr222, Ser272, Thr334 and Thr338 became 32P-labelled in stressed KB cells and labelling was prevented by the specific RK inhibitor SE 203580, establishing that RK phosphorylates Thr25, Thr222, Ser272 and Thr334 in vivo. The 32P-labelling of Thr338 is likely to result from autophosphorylation. GST-MAPKAP kinase-2 lacking the N-terminal domain was inactive, but activated fully when phosphorylated at Thr222, Ser272 and Thr334 by p42 MAPK or RK. In contrast, full-length GST-MAPKAP kinase-2 was phosphorylated at Thr25 (and not activated) by p42 MAPK, suggesting a role for the N-terminal domain in specifying activation by RK in vivo. The mutant Asp222/Asp334 was 20% as active as phosphorylated MAPKAP kinase-2, and this constitutively active form may be useful for studying its physiological roles.

    AB - MAP kinase-activated protein (MAPKAP) kinase-2 is activated in vivo by reactivating kinase (RK), a MAP kinase (MAPK) homologue stimulated by cytokines and cellular stresses. Here we show that in vitro RK phosphorylates human GST-MAPKAP kinase-2 at Thr25 in the proline-rich N-terminal region, Thr222 and Ser272 in the catalytic domain and Thr334 in the C-terminal domain. Using novel methodology we demonstrate that activation of MAPKAP kinase-2 requires the phosphorylation of any two of the three residues Thr222, Ser272 and Thr334. Ser9, Thr25, Thr222, Ser272, Thr334 and Thr338 became 32P-labelled in stressed KB cells and labelling was prevented by the specific RK inhibitor SE 203580, establishing that RK phosphorylates Thr25, Thr222, Ser272 and Thr334 in vivo. The 32P-labelling of Thr338 is likely to result from autophosphorylation. GST-MAPKAP kinase-2 lacking the N-terminal domain was inactive, but activated fully when phosphorylated at Thr222, Ser272 and Thr334 by p42 MAPK or RK. In contrast, full-length GST-MAPKAP kinase-2 was phosphorylated at Thr25 (and not activated) by p42 MAPK, suggesting a role for the N-terminal domain in specifying activation by RK in vivo. The mutant Asp222/Asp334 was 20% as active as phosphorylated MAPKAP kinase-2, and this constitutively active form may be useful for studying its physiological roles.

    KW - Cytokine

    KW - Heat shock

    KW - MAP kinase

    KW - MAPKAP kinase

    KW - Stress

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    M3 - Article

    VL - 14

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    EP - 5930

    JO - EMBO Journal

    JF - EMBO Journal

    SN - 0261-4189

    IS - 23

    ER -

    Ben-Levy R, Leighton IA, Doza YN, Attwood P, Morrice N, Marshall CJ et al. Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2. EMBO Journal. 1995 Jan 1;14(23):5920-5930.