Identification by mass spectrometry of threonine 97 in bovine myelin basic protein as a specific phosphorylation site for mitogen-activated protein kinase.
Bovine myelin basic protein (MBP) was found to be an excellent in vitro substrate (apparent Km = 50 microM) for MAP (mitogen-activated protein) kinase and can be used in lieu of microtubule-associated protein 2 for purification and functional studies of the enzyme. MBP phosphotransferase activity co-purified with MAP kinase during sequential DE52, phenyl-Superose, and gel filtration chromatography, and kinase activities for the two substrates were co-regulated by mitogen stimulation. MAP kinase phosphorylated MBP exclusively on threonine, and only one major phosphopeptide was generated by digestion with trypsin or endoproteinase Lys-C. Using mass spectrometry, we determined that the phosphorylation site is threonine 97, present in the conserved triproline loop of MBP, with (partial) sequence -Thr-Pro-Arg-Thr97-Pro-Pro-Pro-. Thr97 is a known in vivo phosphorylation site in MBP although enzymes capable of phosphorylating this site have not been identified previously. MAP kinase phosphorylated peptide 88-109 from rabbit MBP and a synthetic peptide 91-109 from human MBP but did not phosphorylate either the histone H1 peptide, utilized by p34cdc2, or the peptide substrate for the recently described proline-directed kinase. Thus, the sequence surrounding threonine 97 in bovine MBP may contain essential features of a recognition sequence for MAP kinase.
Erickson
A K
AK
Department of Internal Medicine, University of Virginia, Charlottesville 22908.
Payne
D M
DM
Martino
P A
PA
Rossomando
A J
AJ
Shabanowitz
J
J
Weber
M J
MJ
Hunt
D F
DF
Sturgill
T W
TW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004789:Enzyme Activation; D013058:Mass Spectrometry; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D010769:Phosphothreonine / Q000032:analysis / Q000378:metabolism*; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D008869:Microtubule-Associated Proteins; D004676:Myelin Basic Protein; D010769:Phosphothreonine; D011494:Protein Kinases