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Phosphoproteins Pathway Mapping: Analysis of Multiple Phosphorylation Events in Akt/Pi3‐Kinase Signaling Pathway Using Akt Pathway Array
Author(s) -
Lakkis Montaha,
Knowles Jake,
SCHAEFER ERIK
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.2_supplement.244
Subject(s) - pi3k/akt/mtor pathway , protein kinase b , pten , phosphorylation , signal transduction , microbiology and biotechnology , biology , cancer research , kinase , proto oncogene proteins c akt
Phosphoprotein pathway mapping: Analysis of Multiple Phosphorylation events in Akt/PI3‐Kinase Signaling Pathway Using Akt Pathway Array. Constitutively active PI3K/Akt/mTOR is a survival pathway and a major determinant for cell growth and proliferation in an wide array of human cancers. Mechanisms for its activation include activation and amplification of key signaling targets such as Akt, PI3K and growth factor receptors as well the loss of function of tumor suppressor, PTEN. This pathway is an attractive therapeutic target in cancer and its inhibition provides a new strategy for targeted cancer therapy. Site‐specific phospho‐antibodies are proven to be useful tools to monitor the regulation and signaling cascades of these events and provide critical insight into functional proteomics. In this study, we utilized these antibodies in developing a multiplexing assay using glass‐slides that allows quantitative detection of regulatory phosphorylation sites on 13 markers in the Akt/mTor/AMPK pathways. This assay allows the simultaneous profiling and quantitation of phosphorylation events of a panel of signaling markers in PI3K/Akt/mTOR pathway including tyrosine kinases, substrates and Phosphatases (IR, IGF‐1R, IRS‐1, GSKβ, Glycogen Synthase, Akt, PTEN, PRAS‐40, AMPKα, RSK2, p70S6 Kinase, 4E‐BP1 and STAT3), using minimal amount of tissue or cell lysates. The current Akt PhosphoArray is a rapid, easy to perform assay aims at navigating the complexity of the cellular signaling of key kinases and phosphatases to better understand normal cell functions and determine therapeutic strategies in treating diseases. This multiplex assay allows sensitive and quantitative detection of multiple signaling events across and down the Akt pathway with demonstrated reproducibility, specificity and linearity.