Thr94 in bovine myelin basic protein is a second phosphorylation site for 42-kDa mitogen-activated protein kinase (ERK2).
Treatment of bovine brain myelin basic protein with 42-kDa mitogen-activated protein kinase [p42 MAPK or extracellular signal-regulated kinase 2 (ERK2)] in the presence of ATP and Mg2+ results in phosphorylation of Thr94 and Thr97. Thr94 is not previously known to be an ERK2 phosphorylation site. Both residues are phosphorylated to about the same extent and are in the highly conserved segment Asn91-Ile-Val-Thr94-Pro-Arg-Thr97-Pro-Pro-Pro-Ser101 MALDI mass spectrometry before and after ERK2 treatment revealed the addition of two phosphate groups to the protein. Tryptic cleavage resulted in a single fragment (positions 91-104) carrying the observed mass increase. Tandem mass spectrometry applied to the tryptic peptide showed that both Thr94 and Thr97 are acceptors of phosphate. A singly phosphorylated species could not be detected. Identification of the ERK2 phosphorylation site Thr94 in bovine myelin basic protein reveals a nontraditional phosphate acceptor position, preceded by three noncharged residues (Asn-Ile-Val). Proline at position -2 or -3 from the phosphorylation site, typical for the recognition sequence of proline-directed kinases, is missing. The results provide information for delineation of a further substrate consensus motif for ERK2 phosphorylation.
Hirschberg
Daniel
D
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
Rådmark
Olof
O
Jörnvall
Hans
H
Bergman
Tomas
T
D000818:Animals; D001665:Binding Sites; D001923:Brain Chemistry; D002417:Cattle; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry*; D004676:Myelin Basic Protein / Q000737:chemistry*; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry*; D014357:Trypsin
D004676:Myelin Basic Protein; D010446:Peptide Fragments; D013912:Threonine; D019950:Mitogen-Activated Protein Kinase 1; D014357:Trypsin