Characterization of two functional NKX3.1 binding sites upstream of the PCAN1 gene that are involved in the positive regulation of PCAN1 gene transcription

<p>Abstract</p> <p>Background</p> <p><it>NKX3.1 </it>and <it>PCAN1 </it>are both prostate-specific genes related to prostate development and prostate cancer. So far, little is known about the regulatory mechanisms of the expression of these two genes. In the present study, we found that NKX3.1 upregulated <it>PCAN1 </it>gene transcription in LNCaP prostate cancer cells. To understand the regulatory mechanisms, our work focused on identifying the functional NKX3.1 binding sites upstream of the <it>PCAN1 </it>gene, which might be involved in the positive regulation of <it>PCAN1 </it>expression by NKX3.1.</p> <p>Results</p> <p>We cloned and characterized a 2.6 kb fragment upstream of the <it>PCAN1 </it>gene. Analysis of the 2.6 kb sequence with MatInspector 2.2 revealed five potential binding sites of NKX3.1 transcription factor. Luciferase reporter assays, electrophoretic mobility shift assays, chromatin immunoprecipitation and RNA interference were performed to study the effects of NKX3.1 on <it>PCAN1 </it>gene expression in prostate cancer cells. Our results showed that <it>PCAN1 </it>promoter activity and mRNA expression were increased by transfection with the <it>NKX3.1 </it>containing plasmid (pcDNA3.1-<it>NKX3.1</it>) and that <it>PCAN1 </it>mRNA expression was decreased by RNA interference targeting human <it>NKX3.1 </it>in LNCaP prostate cancer cells. The results of electrophoretic mobility shift assays and chromatin immunoprecipitation showed that NKX3.1 bound to NBS1 (-1848 to -1836) and NBS3 (-803 to -791) upstream of the <it>PCAN1 </it>gene. The luciferase reporter assays showed that NBS1 and NBS3 enhanced the promoter activity in pGL₃-promoter vector with cotransfection of the <it>NKX3.1 </it>containing plasmid. Furthermore, the deletion of NBS1 or both NBS1 and NBS3 reduced <it>PCAN1 </it>promoter activity and abolished the positive regulation of <it>PCAN1 </it>expression by NKX3.1.</p> <p>Conclusion</p> <p>Our results suggested that two functional NKX3.1 binding sites located at -1848 to -1836 and -803 to -791 upstream of the <it>PCAN1 </it>gene were involved in the positive regulation of <it>PCAN1 </it>gene transcription by NKX3.1.</p

Crystal Structure and Substrate Specificity of PTPN12

PTPN12 is an important tumor suppressor that plays critical roles in various physiological processes. However, the molecular basis underlying the substrate specificity of PTPN12 remains uncertain. Here, enzymological and crystallographic studies have enabled us to identify two distinct structural features that are crucial determinants of PTPN12 substrate specificity: the pY+1 site binding pocket and specific basic charged residues along its surface loops. Key structurally plastic regions and specific residues in PTPN12 enabled recognition of different HER2 phosphorylation sites and regulated specific PTPN12 functions. In addition, the structure of PTPN12 revealed a CDK2 phosphorylation site in a specific PTPN12 loop. Taken together, our results not only provide the working mechanisms of PTPN12 for desphosphorylation of its substrates but will also help in designing specific inhibitors of PTPN12