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CDK2-cyclin A |
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- Protein serine/threonine kinase
- Human, recombinant (E. coli)
Description:
CDK2-cyclin A is a member of cyclin-dependent kinases implicated in the eukaryotic cell cycle control. Each phase of the cell cycle is characterized by the expression of different CDK-cyclin complexes that phosphorylate and regulate downstream substrates.
CDK2-cyclin A is a serine/threonine protein kinase composed of the catalytic subunit CDK2 and its positive regulatory subunit cyclin A. For activity, CDK2 requires association with a cyclin and phosphorylation by a CDK-activating kinase (CAK) at a conserved threonine (T160). In vertebrates, CDK2-cyclin A complex is active during S phase of the cell cycle (1).
Recognition Determinants:
The optimal amino acid sequence of substrate for recognition by CDK2, like for other cyclin-dependent kinases (CDKs), is S/TPXR/K, depicting an absolute requirement for Pro at the position +1 and a positively charged residue at the position +3. This data has been defined with respect to the phosphorylation of model peptide substrates. However, the specificity of CDK2-cyclin and other CDKs towards artificial substrates can differ from that of physiological protein substrates in vivo. The distinct cyclin-binding motif termed the Cy motif, with consensus sequence RXL, has been identified in several physiological substrates of CDK2-cyclin A and E. This motif, located C-terminally to the site of phosphorylation, is critical for substrate targeting and serves as a specificity determinant towards different cyclins (2).
Source:
CDK2 phosphorylated at T160 is produced in E. coli by coexpression of human GST-CDK2 (isoform1) and S. cerevisiae GST-Cak1 and purified as an untagged protein. Human cyclin A (residues 173-432) is produced in E. coli as an untagged protein with the N173M mutation (2). All protein coding sequences are cloned under the control of a T7 expression system (kindly provided by Dr. D. Barford) (3).
Reagents Supplied:
CDK2-cyclin A Reaction Buffer (10X)
Enzyme Properties
Protein Kinase Substrate Recognition
Heat Inactivation:
65°C for 20 minutes
Specific Activity:
10,000,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X CDK2-cyclin A Reaction Buffer
Supplemented with 200 μM ATP and 300 μCi/μmol gamma-labeled ATP
Incubate at
30°C.
1X CDK2-cyclin A Reaction Buffer:
50 mM Tris-HCl
10 mM MgCl2
2 mM DTT
1 mM EGTA
0.01 % Brij 35
pH 7.5 @ 25°C
Unit Definition:
One unit is defined as the amount of CDK2-cyclin A required to catalyze the transfer of 1 pmol of phosphate to Histone H1 (10 µM) in 1 minute at 30°C in a total reaction volume of 30 µl.
Concentration:
100,000 units/ml
Storage Conditions:
50 mM HEPES
100 mM NaCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
0.01% Brij 35
pH 7.5 @ 25°C
Storage Temperature:
-70°C
Avoid repeated freeze/thaw cycles.
Notes
General notes:- Molecular Weight: Cdc2 (33 kDa), cyclin A (29 kDa).
- Avoid freeze/thaw cycles. Can be stored for one week or less at -20°C.
- Reaction Conditions: 1X CDK2-cyclin A Reaction Buffer, supplemented with 200 µM ATP and gamma-labeled ATP to a final specific activity of 100-500 µCi/μmol.
Usage notes:- Optimal incubation times and enzyme concentrations must be determined empirically for each particular substrate.
- If possible, the ATP concentration should be at or near saturation (5 -10-fold over Km). Apparent Km values of ATP for most protein kinases are below 100 μM.
However, if the objective is to measure enzyme activity using gamma-labeled ATP, it is best to use 100-200 μM ATP in order to have higher specific activity of gamma-labeled ATP (100-500 cpm/pmol). Also, an excess of substrate should be used, and the level of phosphorylation should not exceed 10% for determination of the initial rate.
To phosphorylate a protein or peptide substrate to completion, the ATP concentration should be about 5-fold over the limited substrate concentration. Higher enzyme concentration and prolonged incubation times should be employed.
Recommended reference:
Protein Phosphorylation: A Practical Approach (1993) ed. Hardie, D.G. IRL Press.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
Contains no detectable protease or phosphatase activities.
Protease Activity:
After incubation of 1,000 units of CDK2-cyclin A with a standard mixture of proteins
for 2 hours at 30ºC, no proteolytic activity could be detected by SDS-PAGE.
Contaminating Phosphatases:
After incubation of 1,000 units of with 50 mM p-nitrophenyl phosphate for 2 hours at 30ºC, no
phosphatase activity could be detected by spectrophotometric analysis.
References
- Morgan, D.O. (1997) Annu. Rev. Cell. Dev. Biol., 13, 261-291.
- Brown, N.R. et al. (1999) Nature Cell Biol., 1, 438-443.
- Stevenson-Lindert, L.M. et al. (2003) J. Biol. Chem., 278, 50956-50960.
Reagents Sold Separately
CDK2-cyclin A Reaction Buffer
Companion Products
Adenosine 5´-Triphosphate (ATP)
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