Cell Signaling Technology

Product Pathways - Glucose Metabolism

SGK3 Kinase #7620

Cell Signaling Technology offers a full line of protein kinases, substrates, antibody detection reagents and HTScan® kits. Browse our "Reagents for High-Throughput Screening" product listing or contact us at drugdiscovery@cellsignal.com.

Description

Purified recombinant full-length human SGK3 kinase, supplied as a GST fusion protein.

Source / Purification

The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing full-length human SGK3 (Met1-Leu496) (GenBank Accession No. NM_013257.3) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.

Gel Staining

Gel Staining

Figure 1. The purity of the GST-SGK3 fusion protein was analyzed using SDS/PAGE followed by anti-GST Western blot (A) or Coomassie stain (B).

Kinase Assay - Radiometric

Kinase Assay - Radiometric

Figure 2. SGK3 kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, 1 µM ATP, 2.5 µg/50 µl PEG20.000, Substrate: GST-GSK3 (a.a. 14-27), variable, and Recombinant SGK3, variable.

Kinase Assay - DELFIA

Kinase Assay - DELFIA

Figure 3. Dose dependence curve of SGK3 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of substrate peptide (#1300) by SGK3 kinase. In a 50 µl reaction, increasing amounts of SGK3 and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)


Quality Control

The theoretical molecular weight of the GST-SGK3 fusion protein is 83 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain and Western blot [Fig.1]. SGK3 kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure SGK3 activity using HTScan® SGK3 Kinase Assay Kit #7621 [Fig.3].

Background

Serum and glucocorticoid-inducible kinase (SGK) is a serine/threonine kinase closely related to Akt (1). SGK is rapidly induced in response to a variety of stimuli, including serum, glucocorticoid, follicle stimulating hormone, osmotic shock and mineralocorticoids. SGK activation can be accomplished via HGF PI3K-dependent pathways and by integrin-mediated PI3K-independent pathways (2,3). Induction and activation of SGK has been implicated in activating the modulation of antiapoptotic and cell cycle regulation (4-6). SGK also plays an important role in activating certain potassium, sodium and chloride channels, suggesting its involvement in the regulation of processes such as cell survival, neuronal excitability and renal sodium excretion (2). SGK is negatively regulated by ubiquitin modification and proteasome degradation (7).

  1. Webster, M.K. et al. (1993) Mol. Cell. Biol. 13, 2031-2040.
  2. Kobayashi, T. and Cohen, P. (1999) Biochem. J. 339, 319-328.
  3. Park, J. et al. (1999) EMBO J. 18, 3024-3033.
  4. Brunet, A. et al. (2001) Mol. Cell. Biol. 21, 952-965.
  5. Mikosz, C.A. et al. (2001) J. Biol. Chem. 276, 16649-16654.
  6. Hayashi, M. et al. (2001) J. Biol. Chem. 276, 8631-8634.
  7. Brickley, D.R. et al. (2002) J. Biol. Chem. 277, 43064-43070.

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Companion Products

This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.

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