Study of the PDK1/AKT Signaling Pathway Using Selective PDK1 Inhibitors, HCS, and Enhanced Biochemical Assays

Alexandra Hofler, Tim Nichols, Stephan Grant, Laura Lingardo, Edward A Esposito, Scott Gridley, Sean T Murphy, John C Kath, Ciarán N Cronin, Michelle Kraus, Gordon Alton, Zhi Xie, Scott Sutton, Mike Gehring, etc.

Anal Biochem. 2011 Jul 15;414(2):179-86.

PMID: 21402045

Abstract:

The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorigenesis. Signal transduction through this pathway requires the assembly and activation of PDK1 and AKT at the plasma membrane. On activation of the pathway, PDK1 and AKT1/2 translocate to the membrane and bind to phosphatidylinositol-(3,4,5)-trisphosphate (PIP(3)) through interaction with their pleckstrin-homology domains. A biochemical method was developed to measure the kinase activity of PDK1 and AKT1/2, utilizing nickel-chelating coated lipid vesicles as a way to mimic the membrane environment. The presence of these vesicles in the reaction buffer enhanced the specific activity of the His-tagged PDK1 (full-length, and the truncated kinase domain) and the activity of the full-length His-tagged AKT1 and AKT2 when assayed in a cascade-type reaction. This enhanced biochemical assay is also suitable for measuring the inhibition of PDK1 by several selective compounds from the carbonyl-4-amino-pyrrolopyrimidine (CAP) series. One of these inhibitors, PF-5168899, was further evaluated using a high content cell-based assay in the presence of CHO cells engineered with GFP-PDK1.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42411778	AKT2, active, His tagged human			Price
IAR4248144	PDK1, active, His tagged human			Price