Cloning and characterization of the promoter region of human focal adhesion kinase gene: nuclear factor kappa B and p53 binding sites.
Focal adhesion kinase (FAK) gene encodes focal adhesion kinase that localizes at contact points of cells with extracellular matrix. It was shown that FAK expression is increased in a variety of malignancies, both at early and advanced stages of tumorigenesis. To understand mechanisms of FAK gene expression and regulation, we cloned and characterized the 5' promoter region of the FAK gene. The 1.2-kb fragment with FAK promoter was placed upstream of the luciferase reporter gene in a pGL3-Basic vector and transfected into different cell lines. Endogenous high-FAK-expressing cell lines showed high levels of luciferase activity in contrast to low-FAK-expressing cells, indicating on transcriptional level of FAK regulation. Serial deletion constructs revealed that a approximately 600 base pair region (-564 to +47) is required for the maximal FAK promoter activity. The 5'-flanking region of FAK is GC-rich and contains several potential transcription factor binding sites, including two NF-kappa B and p53 binding sites. Inhibition of NF-kappa B with NF-kappa B super-repressor decreased FAK luciferase activity. Induction with TNF-alpha increased luciferase activity confirming a role of NF-kappa B transcription factor in the FAK transcriptional activation. The binding of NF-kappa B and p53 transcription factors to the FAK promoter region was demonstrated by electrophoretic mobility shift assay (EMSA). Cotransfection of NF-kappa B and p53 plasmids with FAK promoter luciferase constructs demonstrate induction and inhibition, respectively, of FAK luciferase activity. The results provide a molecular basis for analysis of FAK transcriptional regulation.
Golubovskaya
Vita
V
Department of Surgery, School of Medicine, University of Florida, Gainesville, FL 32610, USA.
Kaur
Aparna
A
Cance
William
W
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D017931:DNA Primers; D016328:NF-kappa B; D016159:Tumor Suppressor Protein p53; D012313:RNA; D008156:Luciferases; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human