Identification of Poly (ADP-ribose) Polymerase-1 (PARP-1) as a Novel Krüppel-like Factor 8-interacting and -regulating Protein

Heng Lu,Xianhui Wang,Tianshu Li,Alison M Urvalek,Lin Yu,Jieli Li,Jinghua Zhu,Qishan Lin,Xu Peng,Jihe Zhao

doi:10.1074/jbc.m110.215632

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.

Highlights

Cellular processes including cell cycle progression (1– 4), oncogenic transformation (5), epithelial-to-mesenchymal transition, migration, and invasion (6)
Inclusion of ethidium bromide in the co-IP reaction did not affect the interaction. These results clearly indicate that Kruppel-like factor 8 (KLF8) directly interacts with Poly(ADP-ribose) polymerase-1 (PARP-1) probably independent of DNA binding in a variety of cell types and that the PARylation of KLF8 by PARP-1 may be important in regulating KLF8 function in the cells
It is plausible to consider that the nuclear localization signal in the zinc finger (ZF) domain serves as a platform for PARP-1 to keep KLF8 in the nucleus after KLF8 is carried into the nucleus by the nuclear importin

Summary

Introduction

Cellular processes including cell cycle progression (1– 4), oncogenic transformation (5), epithelial-to-mesenchymal transition, migration, and invasion (6). PKH3 vector, pKH3-KLF8, or its ZF1,2mCs mutant (KLF8-mut) was transiently transfected into primary PARP-1ϩ/ϩ or PARP-1Ϫ/Ϫ MEF cells.

Results

Conclusion