The human promoter region of JFC1, a phosphatidylinositol 3,4,5-trisphosphate binding ATPase, was isolated by amplification of a 549bp region upstream of the jfc1 gene by the use of a double-PCR system. By primer extension analysis we mapped the transcription initiation site at nucleotide −321 relative to the translation start site. Putative regulatory elements were identified in the jfc1 TATA-less promoter, including three consensus sites for nuclear factor-κB (NF-κB). We analysed the three putative NF-κB binding sites by gel retardation and supershift assays. Each of the putative NF-κB sites interacted specifically with recombinant NF-κB p50, and the complexes co-migrated with those formed by the NF-κB consensus sequence and p50. An antibody to p50 generated a supershifted complex for these NF-κB sites. These sites formed specific complexes with nuclear proteins from tumour necrosis factor α (TNFα)-treated WEHI 231 cells, which were supershifted with antibodies against p50 and p65. The jfc1 promoter was transcriptionally active in various cell lines, as determined by luciferase reporter assays following transfection with a jfc1 promoter luciferase vector. Co-transfection with NF-κB expression vectors or stimulation with TNFα resulted in significant transactivation of the jfc1 promoter construct, although transactivation of a mutated jfc1 promoter was negligible. The expression of a dominant negative IκB (inhibitor κB) decreased basal jfc1 promoter activity. The cell lines PC-3, LNCaP and DU-145, but not Epstein—Barr virus-transformed lymphocytes, showed a dramatic increase in the expression of JFC1 after treatment with TNFα, suggesting that transcriptional activation of JFC1 by the TNFα/NF-κB pathway is significant in prostate carcinoma cell lines.
Abbreviations used: CRE, cAMP response element; EBV, Epstein—Barr virus; IκB, inhibitor κB; IKK, IκB kinase; jfc1-LUC, jfc1 luciferase construct; NF-κB, nuclear factor-κB; PI 3-kinase, phosphoinositide 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; RHD, Rel homology domain; TK, thymidine kinase; TNFα, tumour necrosis factor α.
The nucleotide sequence data reported will appear in GenBank®, EMBL, DDBJ and GSDB Nucleotide Sequence Databases under the accession number AF538054.