Study of the PDK1/AKT signaling pathway using selective PDK1 inhibitors, HCS, and enhanced biochemical assays

被引：37

作者：

Hofler, Alexandra ^{[1
]}

Nichols, Tim ^{[2
]}

Grant, Stephan ^{[1
]}

Lingardo, Laura ^{[1
]}

Esposito, Edward A. ^{[3
]}

Gridley, Scott ^{[3
]}

Murphy, Sean T. ^{[4
]}

Kath, John C. ^{[4
]}

Cronin, Ciaran N. ^{[4
]}

Kraus, Michelle ^{[4
]}

Alton, Gordon ^{[1
]}

Xie, Zhi ^{[1
]}

Sutton, Scott ^{[4
]}

Gehring, Mike ^{[5
]}

Ermoliefff, Jacques ^{[1
]}

机构：

[1] Pfizer, Dept Oncol, San Diego, CA 92121 USA

[2] Pfizer, Dept Drug Safety Res & Dev, San Diego, CA 92121 USA

[3] Blue Sky Biotech, Worcester, MA 01605 USA

[4] Pfizer, Dept Chem, San Diego, CA 92121 USA

[5] Pfizer, Dept External Res Solut CoE, San Diego, CA 92121 USA

来源：

ANALYTICAL BIOCHEMISTRY | 2011年 / 414卷 / 02期

关键词：

PDK1; AKT; Kinase inhibitor; Signal transduction; Template-directed assembly (TDA); PROTEIN-KINASE; AKT; PHOSPHORYLATION; CANCER;

D O I：

10.1016/j.ab.2011.03.013

中图分类号：

Q5 [生物化学];

学科分类号：

070307 [化学生物学];

摘要：

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 ART at the plasma membrane. On activation of the pathway, PDK1 and AICT1/2 translocate to the membrane and bind to phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) through interaction with their pleckstrinhomology 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. (C) 2011 Elsevier Inc. All rights reserved.

引用

页码：179 / 186

页数：8

共 33 条

[1]

Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase B alpha [J].