AKT1 Cellular Phosphorylation Assay Service

AKT1 Cellular Phosphorylation Assay (intracellular kinase activity assay) for compound screening and profiling in intact cells

Target Overview

Target Description

AKT1 is a cytosolic serine-/threonine kinase and a member of PI3 kinase-mediated signaling pathways, regulating cell growth, apoptosis, glycogen synthesis, and other cellular responses through its phosphorylation of downstream substrates. AKT1 activation is triggered by binding of phospholipid and phosphorylation at two key residues: Thr308 by PDK1, and Ser473 by PDK2, now identified as mTOR. Deregulation of AKT1 signaling has been associated with cancer, diabetes, and schizophrenia. Its role in anti-apoptotic and pro-mitotic pathways have made AKT1 a molecular target for anti-cancer therapeutic intervention.

HGNC Reference




Cell Line


Target Expression


Assay Properties

Assay Description

Rat1 cells were transduced with a constitutively active AKT1 mutant (myrAKT1) in which the N-terminal regulatory PH-domain was replaced by a myristoylation signal causing membrane translocation of the kinase. The AKT1 mutant, however, was fused to the N-terminus of a modified estrogen receptor (ER) allowing AKT1 activity only in the presence of estrogen-analogue Hydroxy-Tamoxifen (OHT)[1]. OHT-treatment results in activating phosphorylations at Ser473 and Thr308 in AKT1 which leads to kinase activation and subsequent phosphorylation of direct AKT1 substrate GSK3ß at Ser9 as well as further downstream effects such as proliferation and soft agar growth. GSK3ß phosphorylation in lysates is detected by phospho-GSK3ß-Ser9-specific Sandwich-ELISA. The assay is validated using the broad kinase inhibitor staurosporine (see. Fig.1).
[1] Mirza, AM et al.(2000) Cell Growth Differ 11: 279


Substrate phosphorylation as a readout of intracellular kinase activity via ELISA

Screening Location

Freiburg, Germany

Further Information

More information can be found on our website Cellular Phosphorylation Assay Services.

Reference compound IC50 for AKT1

Reference compound IC50 for AKT1

The broad kinase inhibitor Staurosporine blocks AKT1 and inhibits the cellular phospho-GSK3ß-Ser9 response with highly reproducible IC50 values. The graph shows the result of a representative experiment.