Ikaros interacts in vivo and is a substrate for PP1 phosphatase. A, Ikaros proteins phosphorylated in vivo were immunoprecipitated (IP) with anti-Ikaros-CTS antibodies and incubated in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of PP2A or PP1 phosphatases. B, whole cell lysate from VL3-3M2 cells was mixed with glutathione-agarose beads preincubated with GST (lane 1) or GST-Ikaros (IK) fusion protein (lane 2). The bound proteins were eluted and separated by SDS-PAGE, and the presence of PP1 was detected by Western blot (IB). C, 293T cells expressing Ikaros were labeled in vivo with radioactive orthophosphates (PerkinElmer Life Science), and their whole cell lysates were immunoprecipitated with anti-Ikaros-CTS antibodies using protein A beads. Immunoprecipitates were incubated with phosphatase reaction buffer supplemented with phosphatase inhibitors as indicated: a phosphatase inhibitor mixture (PP_i, lane 1), no phosphatase inhibitor (lane 2), or the PP1-specific inhibitor (Inhib-2) (lane 3). The reactions in all lanes were supplemented with the CK2 kinase inhibitors DRB and heparin. Proteins were separated by SDS-PAGE and visualized by autoradiography. D, whole cell lysates from untransfected 293T cells (lane 1), 293T cells transfected with Ikaros (lanes 2-3), VL3-3M2 cells (lanes 4-6), or other human (lanes 7-9) or murine (lanes 10-11) cell lines were immunoprecipitated with anti-PP1, anti-hemagglutinin (HA), or anti-PP2A antibodies as indicated. The presence of Ikaros in immunoprecipitates was detected by Western blot with anti-Ikaros-CTS antibody.

Interaction of Ikaros and PP1 requires a specific motif. A, wild-type (WT) Ikaros or its corresponding mutants were transfected into 293T cells and labeled in vivo with radioactive [³²P]orthophosphate. The whole cell lysates were immunoprecipitated with anti-Ikaros-CTS antibodies. Immunoprecipitates were incubated with PP1 reaction buffer in the absence or presence of recombinant PP1 as indicated. The samples were resolved by SDS-PAGE and visualized by autoradiography. IK, Ikaros. B, an Ikaros protein map showing deleted segments of the corresponding Ikaros deletion mutants used in experiments above as well the position of the Ikaros point mutant (IK-A465/7) used in other experiments. C, a comparison of PP1-interacting motifs among known PP1 interactors and the Ikaros protein. The amino acids that constitute the core element are presented in bold letters, whereas the contributing amino acids (lysine/arginine proximal to the interacting motif and aspartate/glutamate distal to the interacting motif) are underlined. The consensus sequence of the PP1-interacting motif is at the bottom. D, 293T cells were transfected with wild-type Ikaros or an Ikaros gene that is mutated at the conserved PP1 binding motif (IK-A465/7) as indicated and were in vivo labeled with radioactive [³²P]orthophosphate. Ikaros proteins were immunoprecipitated with anti-Ikaros CTS antibody and incubated in the presence or absence of recombinant PP1 as indicated. After the dephosphorylation reaction, samples were resolved on SDS-PAGE and visualized by autoradiography. E, whole cell lysate from untransfected 293T cells (lane 1) or transfected with wild-type Ikaros (lane 2) or with the IK-A465/7 mutant (lane 3) were immunoprecipitated (IP) with anti-PP1 antibody. Before immunoprecipitation, the whole cell lysates of 293T cells transfected with wild-type Ikaros or the IK-A465/7 mutant were normalized for the presence of Ikaros protein as evidenced by Western blot with anti-Ikaros CTS antibody (top panel). The presence of Ikaros in immunoprecipitates was visualized by Western blot with anti-Ikaros antibodies. The nuclear extract from 293T cells transfected with wild-type Ikaros was used in Western blot as a marker for the presence of Ikaros (lane 4).

The ability of Ikaros to interact with PP1 regulates its DNA binding and subcellular localization. A, nuclear extracts were prepared from 293T cells expressing wild-type or mutant Ikaros (IK) proteins. Equal amounts of Ikaros proteins were used in gel shift reactions as confirmed by Western blot (upper panel). Where indicated, recombinant PP1 (lane 5) or calf intestinal alkaline phosphatase (CIAP, lane 6) were added to the DNA binding reaction. Gel shifts were performed using radiolabeled probes derived from the murine γ satellite A repeat (A) or the murine γ satellite B repeat (B). C, confocal microscopy was used to examine the subnuclear localization of wild-type (WT) and mutant Ikaros proteins. 293T cells that do not express endogenous Ikaros were transduced with wild-type Ikaros (top panel), the Ikaros mutant that is unable to interact with PP1-IK-A465/7 (middle panel), or with a combined Ikaros mutant that is unable to interact with PP1 and is also resistant to phosphorylation by CK2 kinase (IK-A11+A465/7). DNA was visualized by staining with propidium iodine (PI, red), and Ikaros proteins were visualized by anti-Ikaros-CTS antibodies (green). The right panel shows the combined images, with yellow indicating co-localization of Ikaros and DNA.

Dephosphorylation of the Ikaros protein regulates its stability. A, the left panel shows Western blot analysis of 20 μg of whole cell lysate from 293T cells transfected with 5 μg of wild-type Ikaros (IK, lane 1) or 5 μg of Ikaros A465/7 mutant (lane 2) vector. Western blot with anti-β actin antibody was used as a loading control (top panel). The right panel shows a comparison of mRNA levels of wild-type (WT) Ikaros and the Ikaros A465/7 mutant in transduced cells as determined by real time PCR. The relative expression of mutant Ikaros as compared with wild-type Ikaros (assigned a value of 1) is shown. B, schematic representation of the two PEST sequences in the Ikaros protein (bottom panel). The sequence of both PEST sequences is shown (top panel). The amino acids that are known to be phosphorylated by CK2 kinase are marked by asterisk (^*). C, pulse-chase analysis of degradation of wild-type Ikaros (left), the Ikaros-A465/7 mutant (middle), and the Ikaros A11+A465/7 mutant (right) in 293T cells, metabolically labeled with [³⁵S]methionine/[³⁵S]cysteine. Cells were harvested at indicated time points of chase with unlabeled methionine and cysteine. Ikaros proteins were immunoprecipitated with anti-Ikaros-CTS antibody and analyzed by autoradiography. Data shown are from one experiment performed in triplicate (mean ± S.D.) that is representative of five independent experiments. The insets above graphs show the levels of wild-type Ikaros and its mutants as assessed by immunoblotting with anti-Ikaros antibody. D, MOLT-4 cells were treated with cycloheximide to inhibit de novo protein synthesis, and Ikaros expression was determined by Western blot analysis of 20 μg of nuclear extract obtained from untreated MOLT-4 (lanes 1-5), MOLT-4 cells treated with the phosphatase inhibitor calyculin (lanes 6-10), or MOLT-4 cells treated with both calyculin and the CK2-kinase-selective inhibitors DRB (lanes 11-15) or TBB (lanes 16-20). Ikaros proteins were visualized by Western blot using anti-Ikaros-CTS antibody.

Ikaros degradation is mediated by the ubiquitin/proteasome pathway. A-D, whole cell lysates were prepared from 293T cells, as a negative control, or as indicated from untreated MOLT-4 or VL3-3M2 cells or MOLT-4 or VL3-M2 cells that had been treated with the MG132 proteasome inhibitor (MG). Whole cell lysates were immunoprecipitated (IP) with the indicated antibodies, and immunoprecipitates were analyzed by 9% SDS-PAGE followed by immunoblot (IB) with the indicated antibodies. The high molecular weight complexes containing Ikaros (IK)-ubiquitin (Ub) conjugates are marked. The Ikaros detected in MOLT-4 and VL3-3M2 cells in all panels is endogenous Ikaros.

Ikaros targets PP1 into the nucleus and to pericentromeric heterochromatin. A, 293T cells were transfected with wild-type (WT) Ikaros. Localization of transfected Ikaros proteins and endogenous PP1 was examined by immunofluorescence microscopy (top panel) and high magnitude confocal microscopy (bottom panel). Ikaros proteins were detected with anti-Ikaros-CTS antibody (green), whereas endogenous PP1 was detected with anti-PP1 antibody (red). The right panel shows combined images, with yellow indicating co-localization of Ikaros and PP1 proteins. B, 293T cells were transfected with the Ikaros-A465/7 mutant (top panel) or the Ikaros-A11+A465/7 mutant (bottom panel). Confocal microscopy was used to examine localization of transfected Ikaros (IK) proteins and endogenous PP1. Ikaros proteins were detected with anti-Ikaros-CTS antibody (green), whereas endogenous PP1 was detected with anti-PP1 antibody (red). The right panel shows combined images, with yellow indicating co-localization of Ikaros and PP1 proteins. Arrows indicate transfected cells, and arrowheads indicate nontransfected cells within the field in the middle and right panels. C, high magnitude confocal microscopy was used to examine the localization of endogenous Ikaros and endogenous PP1 protein in the Ramos human lymphoma cell line. Endogenous Ikaros proteins were detected with anti-Ikaros-CTS antibody (green), whereas endogenous PP1 was detected with anti-PP1 antibody (red). The right panel shows combined images with yellow indicating co-localization of Ikaros and PP1 proteins.

Proposed regulation of Ikaros activity by CK2 and PP1 signal transduction pathways. The proposed model summarizes the opposing effects of the CK2 kinase and the PP1 pathways on Ikaros function. Our hypothesis is that an optimal balance of these two pathways insures normal cellular proliferation and enables Ikaros to function as a tumor suppressor, whereas increased CK2 kinase activity (as observed in many forms of leukemia) leads to the loss of Ikaros function, increased Ikaros degradation, and malignant transformation.