Identification of poly (ADP-ribose) polymerase-1 (PARP-1) as a novel Kruppel-like factor 8-interacting and -regulating protein.

Krüppel-like factor 8 (KLF8) regulates critical gene transcription and cellular events associated with cancer. However, KLF8-interacting proteins remain largely unidentified. Using co-immunoprecipitation (co-IP), mass spectrometry, and GST pulldown assays, we identified poly(ADP-ribose) polymerase-1 as a novel KLF8-interacting protein. Co-IP and Western blotting indicated that KLF8 is also a substrate. Mutation of the cysteines in the zinc finger domain of KLF8 abolished Pduanyu37-1 interaction. Surprisingly, immunofluorescent staining revealed a cytoplasmic mislocalization of KLF8 in cells or when the interaction was disrupted. This mislocalization was prevented by either Pduanyu37-1 re-expression or inhibition of CRM1-dependent nuclear export. Interestingly, co-IP indicated competition between Pduanyu37-1 and CRM1 for KLF8 binding. Cycloheximide chase assay showed a decrease in the half-life of KLF8 protein when Pduanyu37-1 expression was suppressed or interaction was disrupted. Ubiquitination assays implicated KLF8 as a target of ubiquitination that was significantly higher in Pduanyu37-1(-/-) cells. Promoter reporter assays and chromatin immunoprecipitation assays showed that KLF8 activation on the cyclin D1 promoter was markedly reduced when Pduanyu37-1 was deleted or inhibited or when KLF8-Pduanyu37-1 interaction was disrupted. Overall, this work has identified Pduanyu37-1 as a novel KLF8-binding and -regulating protein and provided new insights into the mechanisms underlying the regulation of KLF8 nuclear localization, stability, and functions.

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