Measurement of Plant Cyclin-Dependent Kinase Activity Using Immunoprecipitation-Coupled and Affinity Purification-Based
Orderly progression of the eukaryotic cell cycle is governed by a coordinated response to intrinsic and extracellular cues through activation of cyclin-dependent kinases (CDKs). It is therefore important to verify the kinase activity of distinct types of
- PDF / 291,053 Bytes
- 14 Pages / 504.567 x 720 pts Page_size
- 100 Downloads / 157 Views
1. Introduction Cyclin-dependent kinases (CDKs) are a family of serine/threonine protein kinases that play important roles in cell-cycle progression in plants as well as yeasts and vertebrates (1, 2). Activation of CDKs requires the binding of a regulatory cyclin subunit and phosphorylation of a conserved threonine residue in the T-loop by CDKactivating kinase (CAK). The activity of CDKs is also regulated by inhibitory phosphorylation and activating dephosphorylation at the ATP-binding sites, threonine 14 and tyrosine 15 (3). Plants have several types of CDKs, two of which, A-type (CDKA) and B-type CDK (CDKB), are principally responsible for cell-cycle N. Dissmeyer and A. Schnittger (eds.), Plant Kinases: Methods and Protocols, Methods in Molecular Biology, vol. 779, DOI 10.1007/978-1-61779-264-9_4, © Springer Science+Business Media, LLC 2011
65
66
H. Harashima and M. Sekine
control (4). CDKA, which includes the canonical PSTAIRE motif, has been implicated in the control of both the G1/S and G2/M transitions (1, 2). CDKB is a plant-specific CDK which shows cell cycle-regulated expression and is mostly active during G2 phase (1, 2). A T-DNA insertional mutant of the single Arabidopsis CDKA gene (CDKA;1) is homozygous lethal because proliferation of generative cells during male gametogenesis is blocked (5, 6). We have used tobacco Bright Yellow-2 (BY-2) cells to evaluate the kinase activities of various types of CDK/cyclin complexes (7–11). BY-2 cells have been extensively studied as a model suspension culture cell, because they are highly homogeneous and their cell cycle can be synchronized up to a high degree with a combination of aphidicolin and propyzamide (12). In addition, the production of recombinant proteins by the baculovirus expression system provides several advantages. First, insect cells have the capability of proteolytic processing such as disulfide bond formation, glycosylation, and phosphorylation of the recombinant protein. Second, multiple proteins can be expressed in this system. Thus, these advantages are useful to analyze which pair of cyclins and CDKs possesses kinase activity in vitro. In this chapter, we describe the method for an immunoprecipitationcoupled kinase assay using suspension culture cells (BY-2) expressing GFP-fused tobacco CDKA and insect cells expressing FLAG-tagged tobacco CDKA.
2. Materials 2.1. Preparation of p13 suc1 Beads
1. Glycerol stock of Escherichia coli expressing p13suc1 protein such as E. coli BL21 (DE3); pET-Suc1. 2. 2× YT medium: Bacto-tryptone 16 g/L, Bacto-yeast extract 10 g/L, NaCl 5 g/L. 3. 3-L Baffled flasks. 4. Isopropyl-b-d-thiogalactopyronoside (IPTG, Nacalai tesque): prepare 1 M stock. Store in aliquots at −20°C. 5. Buffer 1: 20 mM Tris–HCl, 2 mM EDTA, pH 8.0. 6. Buffer 2: buffer 1 containing 1 M NaCl. 7. 50-mL Tubes. 8. Sonicator (such as Astrason XL2020 Ultrasonic Processor using a microtip probe set at 3). 9. 10% (w/v) Polyethylene glycol mono-p-isooctylphenyl ether (Triton X-100). 10. Water bath. 11. FPLC system (GE Healthcare). 12. HiTrap DE
Data Loading...
