Abstract
MAPKAP kinase 2 (MK2) is required for tumor necrosis factor synthesis. Tristetraprolin (TTP) binds to the 3'-untranslated region of tumor necrosis factor mRNA and regulates its fate. We identified in vitro and in vivo phosphorylation sites in TTP using nanoflow high pressure liquid chromatography microelectrospray ionization tandem mass spectrometry and novel methods for direct digestion of TTP bound to affinity matrices (GSH-beads or anti-Myc linked to magnetic beads). MK2Delta3B, activated in Escherichia coli by p38alpha, phosphorylates TTP in vitro at major sites Ser(52) and Ser(178) (>10-fold in abundance) as well as at several minor sites that were detected after enriching for phosphopeptides with immobilized metal affinity chromatography. MK2 phosphorylation of TTP creates a functional 14-3-3 binding site. In cells, TTP was phosphorylated at Ser(52), Ser(178), Thr(250), and Ser(316) and at SP sites in a cluster (Ser(80)/Ser(82)/Ser(85)). Anisomycin treatment of NIH 3T3 cells increased phosphorylation of Ser(52) and Ser(178). Overexpression of MK2 sufficed to increase phosphorylation of Ser(52) and Ser(178) but not Ser(80)/Ser(82)/Ser(85) or Thr(250). Thus, Ser(52) and Ser(178) are putative MK2 sites in vivo. Identified phosphosite(s) may be biologic switches controlling mRNA stability and translation.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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14-3-3 Proteins
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3' Untranslated Regions
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Amino Acid Sequence
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Animals
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Binding Sites
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Cell Line
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Chromatography
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Chromatography, High Pressure Liquid
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Cricetinae
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DNA-Binding Proteins*
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Dose-Response Relationship, Drug
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Glutathione / metabolism
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Glutathione Transferase / metabolism
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Humans
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Immediate-Early Proteins / metabolism*
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Intracellular Signaling Peptides and Proteins
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Kidney / cytology
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Magnetics
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Mice
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Mitogen-Activated Protein Kinase 14
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Mitogen-Activated Protein Kinases / metabolism
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Models, Chemical
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Molecular Sequence Data
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NIH 3T3 Cells
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Peptides / chemistry
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Phosphorylation
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Plasmids / metabolism
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Precipitin Tests
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Protein Binding
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Protein Biosynthesis
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Protein Serine-Threonine Kinases / metabolism
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Protein Serine-Threonine Kinases / physiology*
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RNA, Messenger / metabolism
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Recombinant Fusion Proteins / metabolism
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Serine / chemistry*
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Spectrometry, Mass, Electrospray Ionization
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Time Factors
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Transfection
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Tristetraprolin
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Tyrosine 3-Monooxygenase / chemistry*
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Tyrosine 3-Monooxygenase / metabolism
Substances
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14-3-3 Proteins
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3' Untranslated Regions
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DNA-Binding Proteins
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Immediate-Early Proteins
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Intracellular Signaling Peptides and Proteins
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Peptides
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RNA, Messenger
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Recombinant Fusion Proteins
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Tristetraprolin
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ZFP36 protein, human
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Zfp36 protein, mouse
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Serine
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Tyrosine 3-Monooxygenase
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Glutathione Transferase
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MAP-kinase-activated kinase 2
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Protein Serine-Threonine Kinases
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Mitogen-Activated Protein Kinase 14
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Mitogen-Activated Protein Kinases
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Glutathione