Recombinant full length human SGK3 (serine/threonine-protein kinase 3). This construct contains an N-terminal GST-tag. The recombinant protein was affinity purified and is active.
50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.25 mM DTT, 0.1 mM EGTA, 0.1 mM EDTA, 0.1 mM PMSF, 25% glycerol.
MW
82 kDa
Amino Acids
full length
Specific Activity
90 pmol/min/µg
Genbank #
NM_013257
UniProt #
Q96BR1
Tag(s)
N-terminal GST-tag
Background
SGK3 is a member of the SGK family and encodes a phosphoprotein with a PX (phox homology) domain. PKD1 can phosphorylate and activate SGK3 in vitro (1). A 10-fold increase in PKD1 increases the phosphorylation status of SGK3. When expressed in human embryonic kidney cells, IGF1 and peroxide significantly activate SGK3, and the activation could be reduced by preincubation with inhibitors of PI3 kinase. A yeast 2-hybrid screen found direct interaction between human SGK3 and GSK3β. SGK3 phosphorylates several target proteins and has a role in neutral amino acid transport and activation of potassium and chloride channels (2).
References
1. Kobayashi, T. et al: Characterization of the structure and regulation of two novel isoforms of serum- and glucocorticoid-induced protein kinase. Biochem. J. 344: 189-197, 1999. 2. Gamper, N. et al: K+ channel activation by all three isoforms of serum- and glucocorticoid-dependent protein kinase SGK. Europ. J. Physiol. 445: 60-66, 2002.
Storage/Stability
At least 6 months at -80°C.
Assay Conditions
SGK3 activity was measured by using the Akt (PKB) synthetic peptide (CKRPRAASFAE) diluted in distilled water to a working concentration of 1 mg/ml, in a [33P]-ATP based assay. Reaction was initiated by mixing increasing amounts of SGK3 with 1250 pmoles of [33P]-ATP in 5 mM MOPS, pH 7.2, 2.5 mM β-glycerol-phosphate, 5 mM MgCl2, 1 mM EGTA, 0.4 mM EDTA, 50 ng/µl BSA, 5% glycerol prepared with 50 µM DTT, 50 μM ATP and substrate at a final concentration of 200 µg/ml.
The reaction was initiated by addition of [33P]-ATP Assay Cocktail, followed by a 15-minute incubation at 30°C. The reaction was terminated by spotting the reaction mixture on phosphocellulose P81 paper, air-dry and three 10-minute washes with 1% phosphoric acid solution. Radioactivity was measured in a scintillation counter. The corrected activity (RLU) was calculated by removing the blank value for each sample. The Kinase Specific Activity was calculated as follows: RLU / [(specific activity of [33P]-ATP in cpm/pmol)*(Reaction time in min)*(Enzyme amount in µg or mg)] * [(Reaction Volume) / (Spot Volume)]. The blank was determined from a “no substrate” sample by replacing the substrate solution with an equal volume of distilled water.
Instructions for Use
Thaw on ice and gently mix prior to use. DO NOT VORTEX. Perform a quick spin before opening. Aliquot into small volumes and flash freeze for long term storage. Avoid multiple freeze/thaw cycles.
Applications
Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling.
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