Abstract
Base excision repair (BER) proteins are regulated by post‐translational modifications including phosphorylation. Protein Interacting with Never In Mitosis Gene A‐1 (PIN1) regulates the turnover and function of many phosphoproteins by facilitating the cis‐trans isomerization of phospho‐serine/threonine‐proline motifs. Consequently, we have examined the role of PIN1 in the regulation of BER proteins. Previously, we found that depletion of PIN1 with shRNA in EMT6 murine mammary xenograft tumors increased levels of the BER endonuclease, apurinic/apyrimidinic endonuclease 1 (APE1), compared with control tumors. Here we determined the effect of PIN1 depletion on APE1 expression, subcellular distribution, and activity in EMT6 cells in culture. Cells were transduced with lentivirus encoding small hairpin RNA targeting PIN1, or with an inactive control sequence. Stably transduced cells were selected with puromycin. Cells were lysed, and nuclei and cytosol were separated by centrifugation. Western blotting revealed no difference in total, nuclear, or cytosolic APE1 between the control and knockdown cells. However, APE1 activity, assessed by cleavage of a fluorescently tagged DNA oligomer containing an abasic site, was significantly higher in knockdown compared to control cells (p<0.05). The present results suggest that PIN1 can regulate APE1 function in EMT6 cancer cells.
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