Abstract
BACKGROUND: Ikaros is a DNA-binding protein that acts as master-regulator of hematopoiesis and a tumor suppressor. In thymocytes and T-cell leukemia, Ikaros negatively regulates transcription of terminal deoxynucleotide transferase (TdT), a key protein in lymphocyte differentiation. The signaling pathways that regulate Ikaros-mediated repression of TdT are unknown. Our previous work identified Casein Kinase II (CK2) and Protein Phosphatase 1 (PP1) as regulators of Ikaros DNA binding activity. Here, we investigated the role of PP1 and CK2 in regulating Ikaros-mediated control of TdT expression. PROCEDURES: Ikaros phosphomimetic and phosphoresistant mutants and specific CK2 and PP1 inhibitors were used in combination with quantitative chromatin immunoprecipitation (qChIP) and quantitative reverse transcriptase-PCR (q RT-PCR) assays to evaluate the role of CK2 and PP1 in regulating the ability of Ikaros to bind the TdT promoter and to regulate TdT expression. RESULTS: We demonstrate that phosphorylation of Ikaros by pro-oncogenic CK2 decreases Ikaros binding to the promoter of the TdT gene and reduces the ability of Ikaros to repress TdT expression during thymocyte differentiation. CK2 inhibition and PP1 activity restore Ikaros DNA-binding affinity toward the TdT promoter, as well as Ikaros-mediated transcriptional repression of TdT in primary thymocytes and in leukemia. CONCLUSION: These data establish that PP1 and CK2 signal transduction pathways regulate Ikaros-mediated repression of TdT in thymocytes and leukemia. These findings reveal that PP1 and CK2 have opposing effects on Ikaros-mediated repression of TdT and establish novel roles for PP1 and CK2 signaling in thymocyte differentiation and leukemia.