The identification of raptor as a substrate for p44/42 MAPK

Paul Langlais, Zhengping Yi, Lawrence J. Mandarino

    Research output: Contribution to journalArticle

    9 Citations (Scopus)

    Abstract

    The adaptor protein raptor is the functional identifier for mammalian target of rapamycin (mTOR) complex 1 (mTORC1), acting to target mTOR to specific substrates for phosphorylation and regulation. Using HPLC-electrospray ionization tandem mass spectrometry, we confirmed the phosphorylation of raptor at Ser696, Thr706, Ser721, Ser722, Ser855, Ser859, Ser863, Thr865, Ser877, Ser881, Ser883, and Ser884 and identified Tyr692, Ser699, Thr700, Ser704, Ser854, Ser857, Ser882, Ser886, Ser887, and Thr889 as new, previously unidentified raptor phosphorylation sites. Treatment of cells with insulin increased the phosphorylation of raptor at Ser696, Ser855, Ser863, and Thr865 and suppressed the phosphorylation of Ser722. Ser696 phosphorylation was insensitive to mTOR inhibition with rapamycin, whereas treatment of cells with the MAPK inhibitor PD98059 inhibited the insulin-stimulated phosphorylation of raptor at Ser696. In vitro incubation of raptor with p42 MAPK significantly increased raptor phosphorylation (P < 0.01), whereas phosphorylation of a Ser696Ala mutant was decreased (P < 0.05), suggesting MAPK is capable of directly phosphorylating raptor at Ser696. Mutation of Ser696 to alanine interfered with insulin-stimulated phosphorylation of the mTOR downstream substrate p70S6 kinase. Incubation of cells with the MAPK inhibitor PD98059 and the phosphatidylinositol 3-kinase inhibitor wortmannin decreased the insulin stimulated phosphorylation of raptor, suggesting that the MAPK and phosphatidylinositol 3-kinase pathways may merge at mTORC1.

    Original languageEnglish (US)
    Pages (from-to)1264-1273
    Number of pages10
    JournalEndocrinology
    Volume152
    Issue number4
    DOIs
    StatePublished - Apr 2011

    Fingerprint

    Raptors
    Mitogen-Activated Protein Kinase 3
    Phosphorylation
    Sirolimus
    Phosphatidylinositol 3-Kinase
    Insulin
    Electrospray Ionization Mass Spectrometry
    Mitogen-Activated Protein Kinase 1
    Tandem Mass Spectrometry
    Alanine
    Phosphotransferases

    ASJC Scopus subject areas

    • Endocrinology

    Cite this

    Langlais, P., Yi, Z., & Mandarino, L. J. (2011). The identification of raptor as a substrate for p44/42 MAPK. Endocrinology, 152(4), 1264-1273. https://doi.org/10.1210/en.2010-1271

    The identification of raptor as a substrate for p44/42 MAPK. / Langlais, Paul; Yi, Zhengping; Mandarino, Lawrence J.

    In: Endocrinology, Vol. 152, No. 4, 04.2011, p. 1264-1273.

    Research output: Contribution to journalArticle

    Langlais, P, Yi, Z & Mandarino, LJ 2011, 'The identification of raptor as a substrate for p44/42 MAPK', Endocrinology, vol. 152, no. 4, pp. 1264-1273. https://doi.org/10.1210/en.2010-1271
    Langlais, Paul ; Yi, Zhengping ; Mandarino, Lawrence J. / The identification of raptor as a substrate for p44/42 MAPK. In: Endocrinology. 2011 ; Vol. 152, No. 4. pp. 1264-1273.
    @article{8cd54a0b6bbf480483d6c3c51df80d4b,
    title = "The identification of raptor as a substrate for p44/42 MAPK",
    abstract = "The adaptor protein raptor is the functional identifier for mammalian target of rapamycin (mTOR) complex 1 (mTORC1), acting to target mTOR to specific substrates for phosphorylation and regulation. Using HPLC-electrospray ionization tandem mass spectrometry, we confirmed the phosphorylation of raptor at Ser696, Thr706, Ser721, Ser722, Ser855, Ser859, Ser863, Thr865, Ser877, Ser881, Ser883, and Ser884 and identified Tyr692, Ser699, Thr700, Ser704, Ser854, Ser857, Ser882, Ser886, Ser887, and Thr889 as new, previously unidentified raptor phosphorylation sites. Treatment of cells with insulin increased the phosphorylation of raptor at Ser696, Ser855, Ser863, and Thr865 and suppressed the phosphorylation of Ser722. Ser696 phosphorylation was insensitive to mTOR inhibition with rapamycin, whereas treatment of cells with the MAPK inhibitor PD98059 inhibited the insulin-stimulated phosphorylation of raptor at Ser696. In vitro incubation of raptor with p42 MAPK significantly increased raptor phosphorylation (P < 0.01), whereas phosphorylation of a Ser696Ala mutant was decreased (P < 0.05), suggesting MAPK is capable of directly phosphorylating raptor at Ser696. Mutation of Ser696 to alanine interfered with insulin-stimulated phosphorylation of the mTOR downstream substrate p70S6 kinase. Incubation of cells with the MAPK inhibitor PD98059 and the phosphatidylinositol 3-kinase inhibitor wortmannin decreased the insulin stimulated phosphorylation of raptor, suggesting that the MAPK and phosphatidylinositol 3-kinase pathways may merge at mTORC1.",
    author = "Paul Langlais and Zhengping Yi and Mandarino, {Lawrence J.}",
    year = "2011",
    month = "4",
    doi = "10.1210/en.2010-1271",
    language = "English (US)",
    volume = "152",
    pages = "1264--1273",
    journal = "Endocrinology",
    issn = "0013-7227",
    publisher = "The Endocrine Society",
    number = "4",

    }

    TY - JOUR

    T1 - The identification of raptor as a substrate for p44/42 MAPK

    AU - Langlais, Paul

    AU - Yi, Zhengping

    AU - Mandarino, Lawrence J.

    PY - 2011/4

    Y1 - 2011/4

    N2 - The adaptor protein raptor is the functional identifier for mammalian target of rapamycin (mTOR) complex 1 (mTORC1), acting to target mTOR to specific substrates for phosphorylation and regulation. Using HPLC-electrospray ionization tandem mass spectrometry, we confirmed the phosphorylation of raptor at Ser696, Thr706, Ser721, Ser722, Ser855, Ser859, Ser863, Thr865, Ser877, Ser881, Ser883, and Ser884 and identified Tyr692, Ser699, Thr700, Ser704, Ser854, Ser857, Ser882, Ser886, Ser887, and Thr889 as new, previously unidentified raptor phosphorylation sites. Treatment of cells with insulin increased the phosphorylation of raptor at Ser696, Ser855, Ser863, and Thr865 and suppressed the phosphorylation of Ser722. Ser696 phosphorylation was insensitive to mTOR inhibition with rapamycin, whereas treatment of cells with the MAPK inhibitor PD98059 inhibited the insulin-stimulated phosphorylation of raptor at Ser696. In vitro incubation of raptor with p42 MAPK significantly increased raptor phosphorylation (P < 0.01), whereas phosphorylation of a Ser696Ala mutant was decreased (P < 0.05), suggesting MAPK is capable of directly phosphorylating raptor at Ser696. Mutation of Ser696 to alanine interfered with insulin-stimulated phosphorylation of the mTOR downstream substrate p70S6 kinase. Incubation of cells with the MAPK inhibitor PD98059 and the phosphatidylinositol 3-kinase inhibitor wortmannin decreased the insulin stimulated phosphorylation of raptor, suggesting that the MAPK and phosphatidylinositol 3-kinase pathways may merge at mTORC1.

    AB - The adaptor protein raptor is the functional identifier for mammalian target of rapamycin (mTOR) complex 1 (mTORC1), acting to target mTOR to specific substrates for phosphorylation and regulation. Using HPLC-electrospray ionization tandem mass spectrometry, we confirmed the phosphorylation of raptor at Ser696, Thr706, Ser721, Ser722, Ser855, Ser859, Ser863, Thr865, Ser877, Ser881, Ser883, and Ser884 and identified Tyr692, Ser699, Thr700, Ser704, Ser854, Ser857, Ser882, Ser886, Ser887, and Thr889 as new, previously unidentified raptor phosphorylation sites. Treatment of cells with insulin increased the phosphorylation of raptor at Ser696, Ser855, Ser863, and Thr865 and suppressed the phosphorylation of Ser722. Ser696 phosphorylation was insensitive to mTOR inhibition with rapamycin, whereas treatment of cells with the MAPK inhibitor PD98059 inhibited the insulin-stimulated phosphorylation of raptor at Ser696. In vitro incubation of raptor with p42 MAPK significantly increased raptor phosphorylation (P < 0.01), whereas phosphorylation of a Ser696Ala mutant was decreased (P < 0.05), suggesting MAPK is capable of directly phosphorylating raptor at Ser696. Mutation of Ser696 to alanine interfered with insulin-stimulated phosphorylation of the mTOR downstream substrate p70S6 kinase. Incubation of cells with the MAPK inhibitor PD98059 and the phosphatidylinositol 3-kinase inhibitor wortmannin decreased the insulin stimulated phosphorylation of raptor, suggesting that the MAPK and phosphatidylinositol 3-kinase pathways may merge at mTORC1.

    UR - http://www.scopus.com/inward/record.url?scp=79953206247&partnerID=8YFLogxK

    UR - http://www.scopus.com/inward/citedby.url?scp=79953206247&partnerID=8YFLogxK

    U2 - 10.1210/en.2010-1271

    DO - 10.1210/en.2010-1271

    M3 - Article

    VL - 152

    SP - 1264

    EP - 1273

    JO - Endocrinology

    JF - Endocrinology

    SN - 0013-7227

    IS - 4

    ER -