title: Identification of poly (ADP-ribose) polymerase-1 (PARP-1) as a novel Kruppel-like factor 8-interacting and -regulating protein.
abstract: 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 (PARP-1) as a novel KLF8-interacting protein. Co-IP and Western blotting indicated that KLF8 is also a PARP-1 substrate. Mutation of the cysteines in the zinc finger domain of KLF8 abolished PARP-1 interaction. Surprisingly, immunofluorescent staining revealed a cytoplasmic mislocalization of KLF8 in PARP-1(-/-) cells or when the interaction was disrupted. This mislocalization was prevented by either PARP-1 re-expression or inhibition of CRM1-dependent nuclear export. Interestingly, co-IP indicated competition between PARP-1 and CRM1 for KLF8 binding. Cycloheximide chase assay showed a decrease in the half-life of KLF8 protein when PARP-1 expression was suppressed or KLF8-PARP-1 interaction was disrupted. Ubiquitination assays implicated KLF8 as a target of ubiquitination that was significantly higher in PARP-1(-/-) cells. Promoter reporter assays and chromatin immunoprecipitation assays showed that KLF8 activation on the cyclin D1 promoter was markedly reduced when PARP-1 was deleted or inhibited or when KLF8-PARP-1 interaction was disrupted. Overall, this work has identified PARP-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.
authors.affiliation: Burnett School of Biomedical Sciences, University of Central Florida College of Medicine, Orlando, Florida 32827, USA.
authors.forename: Heng
authors.initials: H
authors.lastname: Lu
authors.forename: Xianhui
authors.initials: X
authors.lastname: Wang
authors.forename: Tianshu
authors.initials: T
authors.lastname: Li
authors.forename: Alison M
authors.initials: AM
authors.lastname: Urvalek
authors.forename: Lin
authors.initials: L
authors.lastname: Yu
authors.forename: Jieli
authors.initials: J
authors.lastname: Li
authors.forename: Jinghua
authors.initials: J
authors.lastname: Zhu
authors.forename: Qishan
authors.initials: Q
authors.lastname: Lin
authors.forename: Xu
authors.initials: X
authors.lastname: Peng
authors.forename: Jihe
authors.initials: J
authors.lastname: Zhao
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D028884:Karyopherins / Q000235:genetics / Q000378:metabolism; D051741:Kruppel-Like Transcription Factors; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers; D000097606:Exportin 1 Protein
chemical_list: D019938:Cyclin D1; D028884:Karyopherins; D051741:Kruppel-Like Transcription Factors; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D018160:Receptors, Cytoplasmic and Nuclear; D012097:Repressor Proteins; D014157:Transcription Factors; D000097606:Exportin 1 Protein; C530104:CCND1 protein, human; C530105:Ccnd1 protein, mouse; C507402:KLF8 protein, human; C475812:Klf8 protein, mouse; C499320:PARP1 protein, human; C499321:Parp1 protein, mouse