mik1+ encodes a tyrosine kinase that phosphorylates p34cdc2 on tyrosine 15.
mik1+ and wee1+ function to regulate the tyrosine phosphorylation of p34cdc2 in Schizosaccharomyces pombe (Lundgren, K., Walworth, N., Booher, R., Dembski, M., Kirschner, M., and Beach, D. (1991) Cell 64, 1111-1122). wee1+ encodes a tyrosine kinase that directly phosphorylates p34cdc2 on tyrosine 15, resulting in the inactivation of the cyclin B/p34cdc2 complex. We have overproduced the mik1+ gene product in insect cells and in S. pombe in order to characterize it biochemically. Immunoprecipitates of Mik1 from both sources catalyzed the phosphorylation of p34cdc2 on tyrosine 15 whereas immunoprecipitates of a kinase-deficient mutant of Mik1 were negative in this assay. Mik1 overproduced in insect cells was partially purified by column chromatography, and column fractions were assayed for their ability to phosphorylate p34cdc2 on tyrosine 15. Two major peaks of Mik1 protein were detected by gel filtration chromatography. One peak eluted in the void volume, and a second peak eluted with an apparent molecular mass expected for monomeric Mik1 (approximately 68 kDa). The tyrosine 15 kinase activity co-eluted with the 68 kDa form of Mik1. These results indicate that mik1+ encodes a tyrosine kinase that directly phosphorylates p34cdc2 on tyrosine 15.
Lee
M S
MS
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Enoch
T
T
Piwnica-Worms
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002460:Cell Line; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029702:Schizosaccharomyces pombe Proteins; D018411:Spodoptera; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; D014443:Tyrosine; C090679:mik1 protein, S pombe; D011505:Protein-Tyrosine Kinases; D016203:CDC2 Protein Kinase