Abstract
The role of post-translational modification, such as sumoylation, in modulating the efficacy of doxorubicin (Dox) treatment remains unclear. Transcriptional cofactor KRAB domain-associated protein 1 (KAP1) has been shown to complex with the KRAB zinc finger protein, ZBRK1, to repress the transcription of target genes. Through a combination of proteomic screening and site-directed mutagenesis approaches, we have identified lysines 554, 779, and 804 as the major sumoylation sites in KAP1. We then present evidence that Dox-mediated induction of cell cycle regulator p21 expression is differentially regulated by KAP1 sumoylation status. Moreover, the KAP1 sumoylation level was transiently decreased upon Dox exposure, and transfection with the KAP1 sumoylation mimetic, SUMO-1-KAP1, desensitizes breast cancer MCF-7 cells to Dox-elicited cell death. The sumoylation-dependent stimulation of KAP1 function is achieved by enhancing the methylation of H3-K9 and attenuating the acetylation of H3-K9 and H3-K14 at the p21 core promoter. We also show that occupancy of ZBRK1 response elements located at the p21 promoter by ZBRK1.KAP1 is independent of KAP1 sumoylation. Hence, sumoylation of KAP1 represses p21 transcription via a chromatin-silencing process without affecting interaction between KAP1.ZBRK1 and DNA, thus providing a novel mechanistic basis for the understanding of Dox-induced de-repression of p21 transcription. Taken together, our results suggest that Dox-induced decrease in KAP1 sumoylation is essential for Dox to induce p21 expression and subsequent cell growth inhibition in MCF-7 cells.
Highlights
The presented studies expanded our understanding of KRAB domain-associated protein 1 (KAP1) biology, as well as perhaps the role of KAP1sumoylation in regulating Dox-induced p21 expression
We report that KAP1 is a sumoylation target, and its sumoylation level is transiently decreased upon the exposure to chemotherapeutic agent Dox
Our findings suggest that de-sumoylation or sumoylation of KAP1 defines the extent to which Dox induces the expression of cell cycle regulator p21 via ZBRK11⁄7KAP1 response element and subsequent breast cancer MCF-7 cell death
Summary
Cell Culture—HEK293 and MCF-7 cells were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and antibiotics at 37 °C under 5% CO2, whereas T47D cells were maintained in RPMI 1640 medium with 10% fetal bovine serum and antibiotics otherwise as the same conditions above. Antibodies—The anti-FLAG-agarose, FLAG peptide, and anti-FLAG mouse monoclonal antibody were purchase from Sigma-Aldrich. FLAG affinity purification was performed with anti-FLAG M2-agarose (Sigma), and nonspecific binding was washed off with twenty bed volumes of radioimmune precipitation assay buffer for three times. Prior to subjecting to mass spectrometric analysis, eluted proteins were digested with sequencing grade-modified trypsin (Promega, Madison, WI) at 100:1 ratio twice, and the digestion mixture was desalted by passing through ZipTip C18 resins. Liquid Chromatography-Ion Trap Mass Spectrometry—Mass spectrometric analysis of the trypsin-digested, sumoylated proteins was performed using a Thermo Finnigan LCQ Deca XP Plus mass spectrometer with reverse phase liquid chromatography implemented with an Ultra Plus II LC system (MicroTech Scientific) using a 150-mm ϫ 75-m C-18 reverse-phase column (5-m, 300-Å particles) from Micro-Tech Scientific. Analysis of MS Spectra—A Beta test version of Bioworks (Bioworks 3.1) on a nine-node (2 central processing units/node)
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