Identification of Poly (ADP-ribose) Polymerase-1 (PARP-1) as a Novel Krüppel-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.

Poly(ADP-ribosyl)ation of p53 induces gene-specific transcriptional repression of MTA1

“…However, much attention should be given to our observation that knockdown or chemical inhibition of PARP1 decreased the 5-FU-induced p53 binding on the promoter of MTA1, and subsequently recovered the expression level of the MTA1 gene (Figures 5c and 6a). Similarly, PARP-1 interacts with and PARylates other transcription factors, such as SMAD3/4 and the Kruppel-like factor 8, thereby regulating their target genes (Lo¨nn et al, 2010;Lu et al, 2011). These observations indicate that inhibition of PARP-1 may cause disturbances in gene expression that may drive the stimulation of a genetic network that enhances oncogenic potential.…”

Implications of Epithelial–Stromal Interaction 1 in Diseases Associated with Inflammatory Signaling

“…Being consistent with the change in the activity of caspase 8, the manipulation of EPSTI1 expression greatly impacts the cleavage of poly(ADP-ribose) polymerase 1 (PARP-1), a hallmark of active apoptosis. 37,54,55 In concert with these findings, some interesting connections can be drawn between the pathways associated with EPSTI1. Primarily, both EPSTI1's upstream KLF8 and downstream NF-κB promote the expression of matrix metalloproteinase 9 (MMP9) and cyclin D1, which are important for cancer invasion and proliferation, respectively, by activating the transcription of the genes in a p300-dependent fashion.…”

“…2), a dual transcription factor implicated in cancer of many types 29,[32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] that promotes the expression of EPSTI1 by transcriptional activation of the gene promoter. 23 Notably, KLF8 has been demonstrated to potently induce EMT by repressing E-cadherin 45 and cancer stem cell traits.…”

“…This revokes the possibility that KLF8 and NF-κB work synergistically to activate their targets, such as cyclin D1 and MMP9. 37 In addition to inhibiting caspase 8, EPSTI1 was also shown to interact with RAC-alpha serine/threonine-protein kinase (AKT1) and breast cancer antiestrogen resistance (BCAR3) protein. 13 AKT is a master player in the cell survival pathways, and BCAR3 plays a role in breast cancer resistance against estrogen-based hormonal therapy.…”

“…41,42,56,57 EPSTI1 inhibition of PARP-1 cleavage is also of interest, given that the nuclear presence of KLF8 depends on its interaction with PARP-1. 37 Notably, PARP-1 binds to p300 to mediate the NF-κB transcriptional activation. 58 However, PARP-1 does not exist with KLF8-p300 complex at the cyclin D1 gene promoter.…”