Measurement of Wee kinase activity.
The Wee kinases (Wee1, Wee2, and Myt1) are major regulators of mitotic entry. They function by phosphorylating Cdc2 and related Cdks on conserved tyrosine and threonine residues. This phosphorylation blocks the activity of the Cdc2 and prevents entry into mitosis. The abundance and activity of the Wee kinases are regulated during the cell cycle and development. In this chapter, we describe several procedures to measure the activity of the Wee kinases found either in crude extracts or in purified preparations. Specific protocols include the production and purification of recombinant Cdc2/Cyclin B substrate, the production of crude subcellular extract fractions, the purification of endogenous or recombinant Wee kinases, Wee kinase assays, and the Histone H1 kinase assay to measure Cdc2 activity. In addition, support protocols are provided that describe the use and production of Ni-IDA beads for the purification of Histidine-tagged proteins, and the use of the baculovirus expression system to produce recombinant proteins.
Mueller
Paul R
PR
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL, USA.
Leise
Walter F
WF
D000818:Animals; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000235:genetics / Q000302:isolation & purification; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002614:Chelating Agents; D019926:Cyclin B / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D007098:Imino Acids; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D009532:Nickel; D009865:Oocytes / Q000166:cytology / Q000201:enzymology; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity; D014981:Xenopus; D029867:Xenopus Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D002614:Chelating Agents; D019926:Cyclin B; D007098:Imino Acids; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D009532:Nickel; D011494:Protein Kinases; C095970:MYT1 kinase, Xenopus; C466028:Wee2 protein, Xenopus; C102333:histone H1 kinase; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; C008109:iminodiacetic acid