Parallel Reaction Monitoring Assays for Phosphorylation Sites of AKT Isoforms in Patient-Derived Breast Cancer Xenografts with PI3K-AKT Pathway Aberrations

摘要

The AKT (Protein Kinase B) family of kinases are involved in multiple cellular processes regulating cell proliferation, survival, and metabolism. The AKT family is comprised of three, highly homologous isoforms (AKT1, 2 and 3) that are the products of three genes (AKT1, chromosome 14; AKT2, chromosome 19; AKT3, chromosome 1) that can regulate independent cellular functions. From studies on mice with gene knockouts of individual AKT genes and knockout combinations, a variety of phenotypes have been reported (reviewed in Hay, 2011). The effects of these knockouts at the individual gene level has not been elucidated. Mutants of AKT can lead to increased disease states such as diabetes due to insulin resistance or the initiation of various cancer types (Hers et al., 2011; Gonzalez and McGraw, 2009; Cicenas, 2008). Although the precise role of each isoform is not well understood in individual tissues, loss- or gain of function mutations within the PI3K/AKT pathway leads to increased incidence of breast tumor development. Notably, loss-of-function of the PTEN phosphatase, was found in 38% of invasive breast cancer tumors (Bose et al., 2002). Figure 1 shows a canonical pathway for AKT signaling. The general paradigm of AKT activation is mediated through the second messenger phosphatidylinositol (3,4,5)-triphosphate (PIP3) which is generated from the activation phosphatidylinositol 3-kinase (PI3K). Subsequent activation of AKT by phosphorylation at Thr308 and Ser473, results in the initiation of downstream signaling components of the PI3K/AKT signaling pathway (Wickenden and Watson, 2010; Hers et al., 2011). The role of phosphorylation of the different AKT isoforms in defining functional specificity is not well understood at the tissue level.