Abstract 935: Protein phosphatase 1 (PP1) regulates tumor suppressor activity of Ikaros in leukemia cells

Abstract

Abstract Ikaros encodes a DNA-binding zinc finger protein that acts as a tumor suppressor in human leukemia. The essential components of Ikaros’ activity include DNA binding and transcriptional repression via chromatin remodeling. We have previously reported that Ikaros directly interacts with Protein Phosphatase 1 (PP1) and undergoes dephosphorylation at specific amino acids (Popescu et al. J Biol Chem 2009 284(20):13869). PP1-mediated dephosphorylation of Ikaros regulates its DNA-binding to probes derived from gamma satellites of pericentromeric heterochromatin, and controls subcellular, pericentromeric localization of Ikaros in vivo. We extended our studies to determine how PP1-mediated dephoshorylation of Ikaros controls Ikaros’ function in regulating the expression of its target genes. An Ikaros mutant that is unable to associate with PP1 (IK 465/7A), and consequently hyperphosphorylated was compared to the wild type Ikaros for: 1) DNA-binding affinity toward the promoters of Ikaros’ target genes; 2) interaction with the NuRD chromatin remodeling complex; and 3) ability to repress Ikaros’ target genes using a luciferase reporter assay. Results showed that the lack of dephosphorylation by PP1 and the resulting increase in phosphorylation of Ikaros is responsible for its inability to bind the promoters of several Ikaros target genes. The absence of interaction with PP1 inhibits binding of Ikaros to the chromatin remodeling complex NuRD. In addition, the inability to interact with PP1 phosphatase abolished Ikaros’ repressor function as evidenced by failure of the IK 465/7A mutant to repress transcription of luciferase in the reporter assay. These results established that dephosphorylation of Ikaros by PP1 is essential for its function as a regulator of gene expression. Studies using an Ikaros mutant that is unable to interact with PP1, but that also contains 11 alanine (phosphoresistant) mutations at CK2 phosphorylation sites (IK 465/7A+A11 mutant) established which of these functional changes are the result of CK2-mediated phosphorylation. Results showed that IK 465/7A+A11 is able to bind to the Ikaros consensus biding sites at promoters of the Ikaros target genes, and to associate with the NuRD remodeling complex. Inhibition of Ikaros phosphorylation by CK2 kinase also restored Ikaros’ ability to repress transcription of target genes in the luciferase reporter assay. Thus, the introduction of 11 alanine mutations at CK2 phosphorylation sites restored the DNA-binding ability and chromatin remodeling function, as well as the transcriptional repressor function of the Ikaros mutant despite its inability to interact with PP1. Our results suggest that dephosphorylation of Ikaros by PP1 and its phosphorylation by CK2 kinase are two opposing mechanisms that control Ikaros’ function in the regulation of gene expression in leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 935. doi:10.1158/1538-7445.AM2011-935