Identification of an autoinhibitory region in the activation loop of the Mos protein kinase.
The Mos protein is a serine/threonine protein kinase which acts to regulate progression through meiosis in vertebrate oocytes. Although Mos function is dependent on its ability to act as a protein kinase, little is known about the factors which regulate Mos kinase activity. To understand the mechanism by which Mos kinase activity is regulated, we have used molecular modeling to construct a three-dimensional model of Mos based on the crystallographic coordinates of cyclic AMP-dependent kinase (PKA). This model identified a loop in Mos which is positioned near the active site and appears capable of blocking substrate access to the active site. Mutagenesis was used to construct altered forms of the Mos protein with deletions of parts or all of the loop. In vitro kinase assays showed that Mos proteins with the loop removed had up to a fourfold increase in kinase activity compared with the wild-type protein, indicating that the loop acts in an autoinhibitory manner for Mos kinase activity. Point mutations were also made on individual residues of the loop which were determined from the molecular model to be capable of reaching the active site. Determination of the kinase activities of these mutants showed that individual mutations in the loop region are capable of either increasing or decreasing kinase activity with regard to the wild-type protein. These data suggest that the loop identified in Mos acts as an autoinhibitor of kinase activity.
Robertson
S C
SC
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, 92093-0367, USA.
Donoghue
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry*; D005260:Female; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017231:Proto-Oncogene Proteins c-mos / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D017231:Proto-Oncogene Proteins c-mos; D017868:Cyclic AMP-Dependent Protein Kinases