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Thr94 in bovine myelin basic protein is a second phosphorylation site for 42-kDa mitogen-activated protein kinase
Daniel Hirschberg1, Olof Rådmark, Hans Jörnvall
1Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
Abstract:
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.
Insights
Extracellular signal-regulated kinase 2 (ERK2) phosphorylates bovine myelin basic protein at Thr94 and Thr97. This study identifies a novel ERK2 phosphorylation site, Thr94, expanding understanding of kinase substrate motifs.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- Myelin basic protein (MBP) is a key component of the myelin sheath in the central nervous system.
- Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 2 (ERK2), play crucial roles in cellular signaling pathways.
- Understanding protein phosphorylation sites is vital for elucidating kinase function and cellular regulation.
Purpose of the Study:
- To investigate the phosphorylation of bovine brain myelin basic protein by ERK2.
- To identify specific phosphorylation sites on MBP targeted by ERK2.
- To characterize the substrate consensus motif for ERK2 phosphorylation.
Main Methods:
- Treatment of bovine MBP with purified ERK2 in the presence of ATP and Mg2+.
- MALDI mass spectrometry to detect mass changes indicative of phosphorylation.
- Tryptic digestion and subsequent tandem mass spectrometry (MS/MS) to identify phosphorylated peptides and residues.
Main Results:
- ERK2 treatment resulted in the phosphorylation of both Thr94 and Thr97 on bovine MBP.
- MALDI-MS confirmed the addition of two phosphate groups.
- Tandem MS analysis of the tryptic peptide (residues 91-104) identified Thr94 and Thr97 as the specific phosphorylation sites.
- Thr94 represents a novel ERK2 phosphorylation site, lacking the typical proline residue at the -2 or -3 position.
Conclusions:
- ERK2 phosphorylates bovine MBP at Thr94 and Thr97.
- The identification of Thr94 expands the known repertoire of ERK2 phosphorylation sites.
- These findings contribute to a refined understanding of the substrate consensus motif for ERK2, highlighting its ability to phosphorylate sites beyond traditional proline-directed motifs.