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mik1+ encodes a tyrosine kinase that phosphorylates p34cdc2 on tyrosine 15
M S Lee1, T Enoch, H Piwnica-Worms
1Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Abstract:
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.
Insights
The mik1+ gene product encodes a tyrosine kinase that directly phosphorylates p34cdc2 on tyrosine 15, regulating cell cycle progression in Schizosaccharomyces pombe.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The wee1+ gene product is a tyrosine kinase regulating p34cdc2 phosphorylation.
- Phosphorylation of p34cdc2 on tyrosine 15 inactivates the cyclin B/p34cdc2 complex, controlling cell division.
Purpose of the Study:
- To biochemically characterize the mik1+ gene product.
- To confirm mik1+ encodes a tyrosine kinase involved in p34cdc2 regulation.
Main Methods:
- Overproduction of mik1+ in insect cells and S. pombe.
- Biochemical assays using immunoprecipitated Mik1 and partially purified protein.
- Gel filtration chromatography to determine molecular mass and assess activity.
Main Results:
- Immunoprecipitates of Mik1 catalyzed p34cdc2 tyrosine 15 phosphorylation.
- Kinase-deficient Mik1 mutants showed no activity.
- Tyrosine 15 kinase activity co-eluted with a 68 kDa form of Mik1.
Conclusions:
- mik1+ encodes a tyrosine kinase.
- This kinase directly phosphorylates p34cdc2 on tyrosine 15.
- mik1+ plays a direct role in regulating p34cdc2 activity.