Characterization of the 5′-flanking region of the human PTK6 gene

PTK6 (also known as Brk) is a non-receptor protein tyrosine kinase, whose mRNA was expressed in the limited normal tissues such as colon and small intestine, and in breast carcinomas and breast cancer cell lines. The 813 bp region upstream from the translation initiation codon, which constitutes a functional promoter of the human PTK6 gene, was progressively deleted and fused to the luciferase reporter gene and transient expression of the resultant constructs was measured upon transfection into a breast carcinoma cell line, T-47D. Comparative analysis of luciferase activity revealed two major regions, -93 to -76 and -702 to -655, important for transcriptional regulation. The proximal -93 to -76 region was found to be essential for the function of the minimal promoter. By primer extension and PCR, it was shown that a PTK6 transcript started at the most 5′ upstream is located around base -104. Therefore, the proximal -93 to -76 region is thought to function as a downstream cis-acting element. Luciferase analysis showed that the distal -702 to -655 region contained at least two cis-acting elements. Gel mobility shift assays with T-47D nuclear extract including competition analyses with consensus and mutant oligonucleotides and supershift analyses with NF-κB and Sp1 antibodies showed that NF-κB binds to the sequence from -706 to -688 and Sp1 binds to the sequence from -688 to -669. This study thus provides the first molecular insights into the transcriptional regulation of the human PTK6 gene.