Characterization of fertilization-modulated myelin basic protein kinases from sea star: regulation of Mapk.
The myelin basic protein (MBP)-phosphorylating enzymes present during maturation and early embryogenesis of the sea star (Pisaster ochraceus) were investigated. The major maturation-activated MBP kinase (p45 Mapk) was molecularly cloned based on tryptic sequence information obtained with the purified enzyme and shown to be highly related to human Erk1 with 76% amino acid identity. Kinase assays and immunoblotting studies revealed that Mapk remained highly active until 12 h post-fertilization (PF), after which it declined. By 4 days PF, Mapk protein was no longer detectable. At 3 h PF, about half of the detectable MBP phosphotransferase activity could be attributed to a 75 kDa protein kinase that was distinct from Mapk. Like Mapk, this protein phosphorylated MBP mostly on threonine residues, but it failed to phosphorylate a peptide (APRTPGGRR) based upon the Thr-97 MAP kinase phosphorylation site in MBP. Rather, it phosphorylated a peptide (AAQKRPSQRTKYLA) patterned after the N-terminus of MBP. Our studies also showed a dramatic increase in MBP phosphotransferase activity occurred by 4 days PF that arose from a third kinase that phosphorylated MBP solely on serine residues. This kinase exhibited the following substrate substrate preference: AAQKRPSQRTKYLA, peptide substrate for S6 kinases (AKRRRLSSLRASTSKSESSQK) > MBP > histone H1 > prota-mine > casein > APRTPGGRR. This kinase was not appreciably affected by addition of phosphatidylserine/diacylglycerol, or the staurosporine analogue Roche Compound 3, but it was partly inhibited by a protein kinase C pseudosubstrate peptide. Gel filtration analysis revealed an apparent molecular mass of 41 kDa for the enzyme. Therefore, at least two novel MBP-phosphorylating enzymes distinct from Mapk are preferentially activated following fertilization and early embryogenesis of the sea star.
Lefebvre
D L
DL
Department of Medicine, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Charest
D L
DL
Yee
A
A
Crawford
B J
BJ
Pelech
S L
SL
D000595:Amino Acid Sequence; D000818:Animals; D002453:Cell Cycle; D002478:Cells, Cultured; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D005306:Fertilization; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000378:metabolism*; D009865:Oocytes / Q000201:enzymology / Q000502:physiology; D010770:Phosphotransferases / Q000502:physiology*; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D013215:Starfish / Q000196:embryology / Q000201:enzymology*; D013997:Time Factors
D004676:Myelin Basic Protein; D010770:Phosphotransferases; D011494:Protein Kinases; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases