The Ikaros gene encodes a zinc finger, DNA-binding protein that acts as regulator of gene transcription and chromatin remodeling[1]. Studies of Ikaros mutant mice have established Ikaros as a master regulator of hematopoiesis[1]. Partial arrest or defects in normal hematopoiesis often lead to aberrant cellular proliferation and leukemia/lymphoma, therefore, it is not surprising that Ikaros knockout mice that lack one copy of Ikaros develop T cell leukemia[2]. The remarkable observation is that these mice developed T cell leukemia with 100% penetrance, and in each case, the leukemic clones arose from cells that had lost the single wild-type Ikaros allele[2]. This suggests an essential role for Ikaros as a tumor suppressor in T cell differentiation. In humans, defects in the Ikaros gene (90% of observed defects involved deletions of one allele, while the remainder involved nonsense or functionally inactivating mutations of a single allele) can result in the production of dominant negative (DN) Ikaros isoforms that act to suppress the function of full-length Ikaros. Ikaros defects have been associated with the development of a variety of hematopoietic malignancies. These include childhood acute lymphoblastic leukemia (ALL)[3,4] infant T-cell ALL[5], adult B cell ALL[6], myelodysplastic syndrome[7], acute myeloid leukemia[8], and adult and juvenile chronic myeloid leukemia[9]. Ikaros defects leading to a loss of Ikaros activity have been detected in 30% of pediatric B-cell ALL, in > 80% of BCR-ABL1 ALL, and approximately 5% of T-cell ALL[10,11]. In addition, defective Ikaros has been identified as a poor prognostic marker for childhood ALL[4,12-14]. A noteworthy observation is that, in almost all primary human leukemia cells in which an Ikaros defect is observed, one wild-type Ikaros copy is retained. These data not only show a strong association between the loss of Ikaros function and the development of human leukemia, but also suggest that even a moderate alteration of Ikaros function (e.g. haploinsufficiency) is sufficient to promote malignant transformation. The aberrant expression of small DN Ikaros isoforms has also been associated with the development of human pituitary adenoma[15]. The current hypothesis is that small Ikaros isoforms act as DN mutants in human cells and their overexpression promotes malignant transformation, while the full-length Ikaros acts as a tumor suppressor.

The function of many proteins is regulated by their phosphorylation status. Protein phosphorylation is a reversible, dynamic process. The balance between phosphorylation states of a protein regulates its overall function. The in vivo phosphopeptide mapping of Ikaros provided the first evidence that Ikaros is phosphorylated at multiple sites[17]. The observation that phosphorylated amino acids within Ikaros are evolutionarily conserved suggests that phosphorylation is an important mechanism regulating Ikaros function. Further phosphopeptide mapping demonstrated that Ikaros phosphorylation sites are very similar in primary thymocytes, in leukemia cells, and in the HEK 293T embryonic kidney carcinoma cell line following transduction or transfection to express Ikaros[17,18]. This suggests that phosphorylation of Ikaros occurs by kinases that are present in multiple tissues and that phosphorylation is an integral feature of Ikaros regulation (Figure ​(Figure11).

A subsequent study has identified four novel evolutionarily conserved CK2 phosphorylation sites located at the N-terminal end of Ikaros (Figure ​(Figure11)[18]. Functional analysis of Ikaros phosphomimetic mutants (where phospho-sites are mutated to aspartate to mimic phosphorylation) and phosphoresistant mutants (where phospho-sites are mutated to alanine to mimic the dephosphorylated state) has revealed that phosphorylation by CK2 affects Ikaros function at many different levels. Phosphorylation of amino acids 13 and 294 results in decreased Ikaros DNA binding affinity for probes that are derived from pericentromeric heterochromatin (PC-HC). In vivo, phosphorylation of the same amino acids causes Ikaros to lose its ability to localize into PC-HC, resulting in diffuse nuclear distribution of Ikaros. These results have provided the first evidence that CK2-mediated phosphorylation regulates not only the ability of Ikaros to bind DNA, but also its subcellular localization and function in chromatin remodeling[18]. We want to emphasize that the cell-cycle-specific phosphorylation of Ikaros that occurs during mitosis is not due to the activity of CK2 because: (1) CK2 is active during G1 and S phases of the cell cycle while this phosphorylation is mitosis-specific; and (2) the CK2 consensus site is well established, and the mitosis-specific phosphorylation of Ikaros occurs at a consensus linker sequence that shows no resemblance to the consensus recognition motif of CK2.

The studies described above were limited in that they examined only the phosphorylation of Ikaros amino acids that are detected in vivo. Phosphopeptide mapping suggests the presence of more phosphorylated amino acids on Ikaros and thus the potential for additional phosphorylation-regulated functions of Ikaros. The discovery that Ikaros is dephosphorylated in vivo by protein phosphatase 1 (PP1), and identification of the PP1-Ikaros interaction site, has provided an opportunity for further studies of the role of phosphorylation in regulating Ikaros function[21]. Ikaros with a mutated PP1 interaction site cannot be dephosphorylated by this enzyme. When this mutant is transfected into HEK 293T cells, its protein binds DNA very poorly compared to wild-type Ikaros. This indicates that a very large percentage of Ikaros undergoes phosphorylation in vivo and that dephosphorylation of Ikaros is essential for its activity. In addition, this mutant is unable to localize to PC-HC, which confirms that phosphorylation controls the subcellular localization of Ikaros. When phosphoresistant mutations of the CK2 phosphorylation sites are introduced into the PP1-nonbinding Ikaros mutant, DNA-binding ability and PC-HC localization is restored to a level similar to that observed for wild-type Ikaros. These data have established that CK2-mediated phosphorylation is the major regulator of Ikaros function as a DNA-binding protein and in chromatin remodeling/PC-HC localization[21]. They also suggest that PP1 has the opposite effect on Ikaros function, and that these two signaling pathways are the major regulators of Ikaros activity.

Studies of Ikaros phosphorylation have provided a partial answer to the question concerning the regulation of Ikaros function in normal and leukemia cells, as well as how alteration in the regulation of Ikaros might contribute to leukemia. The studies described above have established phosphorylation/dephosphorylation as one of the major mechanisms that controls Ikaros activity. These data strongly suggest that CK2 is the principal regulator of Ikaros function. CK2 is an extensively studied regulator of cellular proliferation and a tumor-promoter protein[29]. Overexpression of CK2 has been associated with the development of various types of tumors, including mammary gland, prostate, lung, and kidney cancers[30]. Forced expression of the catalytic subunit of CK2 in transgenic mice leads to the development of T-cell leukemia and lymphoma[31-34], similar to that observed in mice with impaired Ikaros function[2,16,35,36]. Thus, we hypothesize that Ikaros function is controlled by its phosphorylation status and that overexpression of CK2 leads to hyperphosphorylation of Ikaros, which results in its loss of tumor suppressor activity and the subsequent development of leukemia (Figure ​(Figure2).2). Additional studies utilizing Ikaros phosphomimetic and phosphoresistant mutants in mouse models and in primary human and murine cells are necessary to confirm and/or refine this hypothesis. The abundance of Ikaros phosphorylation sites, as well as the lack of a complete understanding of Ikaros function in chromatin remodeling and tumor suppression, suggests that many additional signal transduction pathways that regulate Ikaros function remain to be discovered.