Nanog and FAK were shown to be overexpressed in cancer cells. In this report, 
the Nanog overexpression increased FAK expression in 293, SW480, and SW620 
cancer cells. Nanog binds the FAK promoter and up-regulates its activity, 
whereas Nanog siRNA decreases FAK promoter activity and FAK mRNA. The FAK 
promoter contains four Nanog-binding sites. The site-directed mutagenesis of 
these sites significantly decreased up-regulation of FAK promoter activity by 
Nanog. EMSA showed the specific binding of Nanog to each of the four sites, and 
binding was confirmed by ChIP assay. Nanog directly binds the FAK protein by 
pulldown and immunoprecipitation assays, and proteins co-localize by confocal 
microscopy. Nanog binds the N-terminal domain of FAK. In addition, FAK directly 
phosphorylates Nanog in a dose-dependent manner by in vitro kinase assay and in 
cancer cells in vivo. The site-directed mutagenesis of Nanog tyrosines, Y35F and 
Y174F, blocked phosphorylation and binding by FAK. Moreover, overexpression of 
wild type Nanog increased filopodia/lamellipodia formation, whereas mutant Y35F 
and Y174F Nanog did not. The wild type Nanog increased cell invasion that was 
inhibited by the FAK inhibitor and increased by FAK more significantly than with 
the mutants Y35F and Y174F Nanog. Down-regulation of Nanog with siRNA decreased 
cell growth reversed by FAK overexpression. Thus, these data demonstrate the 
regulation of the FAK promoter by Nanog, the direct binding of the proteins, the 
phosphorylation of Nanog by FAK, and the effect of FAK and Nanog 
cross-regulation on cancer cell morphology, invasion, and growth that plays a 
significant role in carcinogenesis.