Characterization of the PKB/AKT Pathway in the Role of Tumorigenesis and Transformation of MCF10A Human Mammary Epithelial Cells

摘要

The Akt homology domain (AM) of Akt has been identified as a potential dimerization site within the kinase, and potentially could serve as a selective inhibitor of these kinases. We fused the AM domains of Akt1 (AH1) and Akt2 (AH2) to EGFP and performed expression studies with these constructs. Overexpression of both in PTEN deficient U87 cells sensitized these cells to apoptosis from camptothecin treatment, while neither was able to do this in PTEN+ MCF-7 cells. AH2 was able to block overexpressed Aktl and endogenous Akts inhibition of AFX as measured by an AFX reporter assay, but not when Akt2 was overexpressed. AFX disinhibition in U87 and MCF-7 cells patterned their effects in the cytotoxicity assay. Finally, we were able to demonstrate that AH2 bound to Akt1 and Akt2, but AHl only bound to Akt1 using co-immunoprecipitation assays; both bound to PDK1. Binding of AH domains to Akt decreased phosphorylation within the activation loop of Akts. We hypothesize that the AH2 domain binds to the AM domain within Akts, and this blocks access either to 3'OH-phosphoinositides or PDK1, and thereby inhibits Akt activation. We further hypothesize that PTEN deficient cells are more susceptible to Akt inhibition PTEN expressing cells.