Identification of in vivo phosphorylation sites of MLK3 by mass spectrometry and phosphopeptide mapping.
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.
Vacratsis
Panayiotis O
PO
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Phinney
Brett S
BS
Gage
Douglas A
DA
Gallo
Kathleen A
KA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D002460:Cell Line; D005591:Chemical Fractionation; D002845:Chromatography / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods*; D010748:Phosphopeptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11
D020930:MAP Kinase Kinase Kinases; D010446:Peptide Fragments; D010748:Phosphopeptides; D014357:Trypsin; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11