Tu1995 The Inhibition of MDSC and M2 Macrophage by Metformin Is Mediated by AMPK-mTOR and Cholesterol Metabolism Pathway

Abstract

inhibit metastasis. We first confirmed direct Akt1-FAK molecule-molecule interaction and measured its strength using an atomic force microscopic. A reflective cantilever was functionalized with either Akt1 or FAK, allowed to bind to a surface coated with the reciprocal molecule, and then the bound pair was physically pulled apart. By measuring the bend in the cantilever, via the deflection of a laser beam, we demonstrated that Akt1 and FAK bind with a force of 50-60 pN, a relatively weak interaction that should be inhibitable. Coimmunoprecipitation of Akt1 with serially truncated FAK constructs demonstrated that Akt1 binding could be achieved with a 33 amino acid segment of the FAK N-terminal region located in the F1 subdomain of its four-point-one, ezrin, radixin, moesin (FERM) domain. Interestingly, this binding region, located between amino acids 94-126, does not contain the Akt1 phosphorylation targets S517/601/695 or the FAK autophosphorylation site Y397. Reciprocal binding experiments showed co-immunoprecipitation of FAK by Akt1 to depend on the catalytic domain of Akt1. In co-immunoprecipitation assays, addition of the synthesized peptide version of the 33 amino acid FAK truncation sequence inhibited binding of Akt1 to the wild type FAK F1 subdomain. Pharmacologic applications of the 33 amino acid peptide were explored in Caco-2 cells transfected with plasmids containing a GFP-conjugated analog of the peptide. Overexpression of this peptide prevented the stimulation of cell adhesion by pressure. Further exploration of this 33 amino acid sequence suggested that Akt1 binding of the wild type FAK F1 subdomain could be modulated by altering the region of its 3-10 helix. A P117S mutation in the F1 subdomain reduced FAK binding to Akt1 whereas a L113A/P116C/P117G triple mutant increased Akt1 binding. Structures analogous to this FAK-derived peptide may prove useful to inhibit cancer metastasis.