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Identification of the sites in myelin basic protein that are phosphorylated by meiosis-activated protein kinase
J S Sanghera1, R Aebersold, H D Morrison
1Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
Abstract:
Myelin basic protein serves as a convenient substrate for detection of a 44 kDa protein-serine/threonine kinase (p44mpk) that is activated near the time of germinal vesicle breakdown in maturing echinoderm and amphibian oocytes. In vitro phosphorylation by purified p44mpk from sea star oocytes was primarily on threonine residues on a single tryptic peptide of bovine brain myelin basic protein. Amino acid composition analysis of the isolated posphopeptide revealed that it was rich in proline residues. Automated solid-phase sequencing by Edman degradation identified the major site as Thr-97 in the sequence NIVTPRTPPPSQGK, which corresponds to residues 91-104 in bovine brain myelin basic protein. Thr-94 was also phosphorylated by p44mpk to a very minor extent.
Insights
A novel protein-serine/threonine kinase (p44mpk) phosphorylates myelin basic protein during oocyte maturation. The primary target site was identified as threonine-97 in bovine brain myelin basic protein.
Area of Science:
- Biochemistry
- Molecular Biology
- Developmental Biology
Background:
- Protein kinases play crucial roles in cellular signaling pathways.
- Oocyte maturation involves complex regulatory mechanisms, including protein phosphorylation.
- Myelin basic protein is a well-characterized substrate in biochemical studies.
Purpose of the Study:
- To identify and characterize a protein-serine/threonine kinase (p44mpk) involved in oocyte maturation.
- To determine the specific phosphorylation sites on myelin basic protein (MBP) targeted by p44mpk.
- To investigate the role of p44mpk in the maturation of echinoderm and amphibian oocytes.
Main Methods:
- In vitro kinase assays using purified p44mpk from sea star oocytes.
- Phosphorylation of bovine brain myelin basic protein as a substrate.
- Tryptic peptide mapping and amino acid composition analysis.
- Automated solid-phase sequencing (Edman degradation) for phosphopeptide identification.
Main Results:
- Purified p44mpk phosphorylated myelin basic protein primarily on threonine residues.
- A single tryptic peptide was identified as the major phosphorylation site.
- Amino acid analysis revealed the phosphopeptide was rich in proline residues.
- Automated sequencing identified Thr-97 as the major phosphorylation site within the sequence NIVTPRTPPPSQGK (residues 91-104 of MBP).
- Thr-94 was identified as a minor phosphorylation site.
Conclusions:
- p44mpk is a novel kinase activated during oocyte maturation.
- Thr-97 of myelin basic protein is a major phosphorylation site for p44mpk.
- The proline-rich sequence surrounding Thr-97 is important for p44mpk recognition and activity.
- p44mpk may play a regulatory role in the events leading to germinal vesicle breakdown during oocyte maturation.