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  • Glycogen Synthase Kinase 3 (GSK-3)

    Description

    Glycogen Synthase Kinase 3 (GSK­‑3) is a serine/threonine protein kinase and one of several protein kinases, which phosphorylate glycogen synthase. It is also called Factor A (FA) for its ability to activate the MgATP-dependent form of the protein phosphatase PP1 called FC (1-4). Recent studies demonstrate that GSK-3 can autophosphorylate Ser, Thr and Tyr. Ser/Thr phosphorylation causes inactivation, and Tyr phosphorylation results in increased activity (Y216 for GSK-3β). GSK-3 expressed in E. coli or insect cells is extensively phosphorylated on Tyr. Molecules lacking phosphate at this position can autophosphorylate after incubation with Mg2+ and ATP. GSK-3 phosphorylates several exogenous substrates, but not on Tyr residues (5,6). 

    • Protein serine/threonine kinase
    • Rabbit skeletal muscle, recombinant (E. coli)
    • Supplied with 10X Reaction Buffer

    Product Source

    Isolated from a strain of E. coli that carries a clone expressing GSK-3β derived from a rabbit skeletal muscle cDNA library (kindly provided by Dr. P. J. Roach) (5).

    Recognition Determinant

    The substrate specificity of GSK-3 is unique and substrate dependent. For some substrates, prior phosphorylation of the substrate to form the motif S/TXXXpS/pT is a strict requirement whereas in other substrates, no previous phosphorylation is needed. In either case, many of the GSK-3 sites have Pro residues close to the modified Ser or Thr (5,7).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    GSK-3 Reaction Buffer-2010X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of GSK-3 required to catalyze the transfer of 1 pmol of phosphate to CREB Phosphopeptide, KRREILSRRPpSYR (400 µM), in 1 minute at 30°C in a total reaction volume of 25 µl.

    Reaction Conditions

    1X GSK-3 Reaction Buffer
    Supplement with 200 μM ATP
    Incubate at 30°C

    1X GSK-3 Reaction Buffer:
    20 mM Tris-HCl
    10 mM MgCl2
    5 mM DTT
    pH 7.5 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    20 mM Tris-HCl
    50 mM NaCl
    5 mM DTT
    1 mM EDTA
    50% Glycerol
    0.03% Brij 35
    pH 7.5 @ 25°C

    Heat Inactivation

    No

    Molecular Weight

    Theoretical: 47 kDa

    Quality Control

    Quality Assurance Statement

    • GSK-3 contains no detectable protease or phosphatase activities.

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Phosphatase Activity (PNPP):
      The product is tested in a reaction containing a p-nitrophenyl phosphate (PNPP), a chromogenic substrate for most phosphatases. After incubation there is no detectable phosphatase activity as determined by spectrophotometric analysis of released p-nitrophenol at 405 nm.
    • Protease Activity (SDS-PAGE):

      The product is tested for protease activity by incubation with a standard mixture of proteins resulting in no detectable degradation of the proteins as determined by SDS-PAGE.

    Notes

    1. Optimal incubation times and enzyme concentrations must be determined empirically for each particular substrate.
    2. 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.
    3. If the source of protein to be phosphorylated is a crude extract of cells or tissue, it is very important to include the appropriate protease and protein phosphatase inhibitors in the lysis buffer and to use shorter incubation time for phosphorylation.

    References

    1. Embi, N., Rylatt, D.B. and Cohen, P. (1980). Eur. J. Biochem.. 107, 519-527.
    2. Hemmings, B.A., Yellowlees, D., Kernohan, J.C. and Cohen, P. (1982). Eur. J. Biochem.. 119, 443-451.
    3. Vandenheede, J.R., Yang, S.D., Goris, J. and Merlevede, W. (1980). J. Biol. Chem.. 255, 11768-11774.
    4. Woodgett, J. R. (1990). EMBO J.. 9, 2431-2438.
    5. Wang, Q.M., Fiol, C.J., DePaoli-Roach, A.A. and Roach, P.J. (1994). J. Biol. Chem.. 269, 14566-14574.
    6. Cole, A. et al. (2004). Biochem. J. . 377, 249-255.
    7. Frame, S. and Cohen P. (2001). Biochem. J.. 359, 1-16.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. How much Glycogen Synthase Kinase 3 (NEB# P6040) should be used?
    2. What is the consensus sequence for GSK-3 (P6040)?
    3. When running a gel, why do I see a band at approximately 90 kDa when the molecular weight of GSK III is 47 kDa?

    Selection Tools

    Make sure to use the appropriate protease and protein phosphatase inhibitors in the lysis buffer and to use shorter incubation time for phosphorylation if the substrate is a crude lysate.
    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.
    1-2 µl of enzyme is a good starting point for a 1 hr incubation of 1µg of protein.
    The consensus sequence is substrate-dependent. In some target proteins, prior phosphorylation is required, but a general recognition sequence has not yet been elucidated