Identification of in Vivo Phosphorylation Sites of MLK3 by Mass Spectrometry and Phosphopeptide Mapping
| Autor: | Brett S. Phinney, Kathleen A. Gallo, Panayiotis O. Vacratsis, Douglas A. Gage |
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| Rok vydání: | 2002 |
| Předmět: |
Phosphopeptides
Spectrometry Mass Electrospray Ionization Resolution (mass spectrometry) Molecular Sequence Data Peptide Chemical Fractionation Mass spectrometry Peptide Mapping Biochemistry High-performance liquid chromatography Cell Line Humans Trypsin Amino Acid Sequence Phosphorylation Protein kinase A Chromatography High Pressure Liquid chemistry.chemical_classification Chromatography Chemistry Phosphopeptide Hydrolysis MAP Kinase Kinase Kinases Precipitin Tests Peptide Fragments A-site Amino Acid Substitution Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Electrophoresis Polyacrylamide Gel |
| Zdroj: | Biochemistry. 41:5613-5624 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi016075c |
| Popis: | MLK3 is a serine/threonine protein kinase that functions as an upstream activator of the JNK pathway. Previous work has suggested that MLK3 is a multiphosphorylated protein. In this study, mass spectrometry coupled with comparative phosphopeptide mapping was used to directly characterize MLK3 in vivo phosphorylation sites. Various types of mass spectrometry were used to analyze MLK3 tryptic peptides separated by C18 reverse-phase HPLC, leading to the identification of Ser(524), Ser(654), Ser(705), Ser(740), Ser(758), Ser(770), Ser(793), and a site found on peptide Ser(11)-Arg(37) within a Gly-rich region as MLK3 phosphorylation sites. Additionally, porous graphitic carbon chromatography successfully retained and resolved phosphopeptides that had eluted along with nonvolatile salts and buffers in the flowthrough fractions from the C18 column. Following resolution by PGC chromatography, MALDI-MS in conjunction with alkaline phosphatase treatment identified Ser(555), Ser(556), Ser(724), and Ser(727) as sites of phosphorylation on MLK3. A proline residue immediately follows 7 of the 11 unambiguously identified phosphorylation sites, suggesting that MLK3 may be a target of proline-directed kinases. Finally, two-dimensional phosphopeptide mapping confirmed that phosphorylation of Ser(555) and Ser(556) of MLK3 is induced by the activated small GTPase Cdc42. |
| Databáze: | OpenAIRE |
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