title: Role of protein phosphorylation in the maturation-induced activation of a myelin basic protein kinase from sea star oocytes.
abstract: We have previously described the purification of a myelin basis protein (MBP) kinase from maturing sea star oocytes (Sanghera, J. S., Paddon, H. B., Bader, S. A., and Pelech, S. L. (1990) J. Biol. Chem. 265, 52-57). The ability of the purified 44-kDa protein to bind azido-ATP and undergo autophosphorylation on the serine residue implied that it is a protein kinase. Furthermore, partial amino acid sequence data has revealed that it is a novel protein kinase, which we have provisionally designated p44mpk. Autophosphorylation of p44mpk to 0.7 mol of phosphate/mol of enzyme was correlated with a modest (approximately 17%) increase in the MBP-phosphorylating activity of the kinase. Rabbit polyclonal antibody raised against purified p44mpk recognized on immunoblots the protein in highly purified preparations as well as crude oocyte extracts. The affinity-purified anti-p44mpk antibody could immunoprecipitate active kinase, but a subpopulation of the antibody also appeared to be inhibitory. Using this antibody, we have demonstrated that the up to 12-fold stimulation of the cytosolic MBP-phosphorylating activity of this kinase that occurs during sea star oocyte maturation is not due to an increase in the amount of enzyme protein, either from a redistribution within the oocyte or protein synthesis. A slight retardation of the migration of the activated p44mpk on sodium dodecyl sulfate-polyacrylamide gels and its tighter interaction with a MonoQ column is consistent with phosphorylation of the kinase during maturation. p44mpk underwent enhanced phosphorylation when oocytes prelabeled with [32P]orthophosphate were induced to mature with 1-methyladenine. The stimulated MBP-phosphorylating activity of p44mpk in cytosols from maturing oocytes was partly stabilized by the presence of the phosphatase inhibitor beta-glycerol phosphate. Furthermore, treatment of purified p44mpk with protein phosphatase 2A and alkaline phosphatase resulted in 56 and 86% decreases, respectively, in the activity of the kinase. Together, these findings strongly implicate a role for phosphorylation of p44mpk in its activation during sea star oocyte maturation.
authors.affiliation: Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
authors.forename: J S
authors.initials: JS
authors.lastname: Sanghera
authors.forename: H B
authors.initials: HB
authors.lastname: Paddon
authors.forename: S L
authors.initials: SL
authors.lastname: Pelech
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D005260:Female; D038362:Glycogen Synthase Kinase 3; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009865:Oocytes / Q000201:enzymology / Q000502:physiology*; D010446:Peptide Fragments / Q000302:isolation & purification; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D013215:Starfish
chemical_list: D010446:Peptide Fragments; D010750:Phosphoproteins; D011494:Protein Kinases; D038362:Glycogen Synthase Kinase 3