Figure 1.
Co-localization and Physical Interactions of Native Ikaros and BTK Proteins in Human Cells.
[A] Nuclear co-localization of Native IK and BTK. ALL-N1 cells were fixed and stained with polyclonal rabbit anti-IK1 (primary Ab)/Alexa Fluor 568 F(ab')2 fragment of goat anti-rabbit IgG (secondary Ab) (red) and mouse anti-BTK MoAb (primary Ab)/Alexa Fluor 488 goat anti-mouse IgG (secondary Ab) (green) antibodies. Nuclei were stained with blue fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI). MERGE panels depict the merge three-color confocal image showing co-localization of IK1 and BTK in DAPI-stained nucleus as magenta immunofluorescent foci (System magnification: 315×). Representative foci of colocalization are indicated with white arrowheads. [B] Co-immunoprecipitation of Native IK and BTK. B1 depicts the results of the BTK Western blot analysis of the IK and BTK immune complexes immunoprecipitated (IP) from ALL-N1 cells. B.2 depicts the results of the IK Western blot analysis of the BTK and IK immune complexes from the same cells. Controls included immunoprecipitations performed without using a primary (10) antibody.
Figure 2.
BTK Expression Levels Control Nuclear Localization of Ikaros.
[A] Cells were stained with mouse monoclonal antibody (mAb) against human BTK and an anti-IK mouse mAb, which was raised against murine IK and which cross-reacts with human IK. TOTO-3 was used for nuclear staining. Depicted are the confocal two-color (green/blue: BTK/TOTO-3 and IK/TOTO-3) merge images of abundantly BTK+ (high BTK) leukemic cells from a pediatric B-precursor ALL case vs. leukemic cells with low BTK expression level from an infant B-precursor ALL case. Nuclear staining for IK was observed in the confocal fluorescence images of primary leukemic B-cell precursors with high BTK expression level. By contrast, leukemic cells with low BTK expression levels showed an aberrant, predominantly cytoplasmic localization of IK (System Magnification: 500×). [B] The DT-40 cell line and its subclones were stained with the rabbit polyclonal, H-100 (sc-13039) antibody against the N-terminus of IK. Depicted are the confocal two-color (red/blue) IK/DAPI merge images of wildtype (WT) DT40 cells, BTK-deficient DT40 cells, and BTK-deficient DT40 cells reconstituted with wildtype btk. The contour of the DAPI-stained nuclei (blue) was marked with a dotted line and shows a significant amount of IK protein (red) outside the nucleus of the BTK− DT40 cells. [C1] Upper panel: Depicted are the confocal single-color and two-color merge images of control Hek293T cells stained with the secondary goat anti-mouse (GAM) antibody and DAPI. No false positive green fluorescence was detected (System Magnification: 630×). Lower panel: Depicted are the confocal single-color and two-color (green/blue: BTK/DAPI) merge images of test Hek293T cells stained with anti-BTK/goat-anti-mouse (GAM) antibody combination and DAPI. BTK (green fluorescence) was localized primarily in the cytoplasm of Hek293T cells around the DAPI-stained blue nucleus. [C2] Left panel: Merge confocal images of untreated control Hek293T cells two-color stained with anti-BTK/goat-anti-mouse (GAM) antibody combination and DAPI. Right panel: Merge confocal images of BTK-siRNA transfected Hek293T cells two-color stained with anti-BTK/goat-anti-mouse (GAM) antibody combination and DAPI 72 h post-transfection. (System Magnification: 630×). BTK depletion was confirmed by Western blot analysis (see Figure 3) [D] Confocal images of Hek293T cells expressing the HA-tagged mutant IK proteins D6 or A12 stained with the mouse monoclonal anti-HA antibody (HA-probe F-7)(primary Ab)/Alexa Fluor 488 goat anti-mouse IgG (secondary Ab) (green) antibody and blue fluorescent DNA dye 4′, 6-diamidino-2-phenylindole (DAPI) following treatment with BTK siRNA (50 nM) vs. PBS (CON). (System Magnification: 630×).
Figure 3.
BTK Expression Levels Control DNA Binding Activity and Transcription Factor Function of Ikaros.
[A1] BTK Western Blot Analysis of RAJI and Hek293T cells. RAJI cells expressed predominantly the 77 kDa isoform of BTK, whereas Hek293T cells expressed predominantly a 65 kDa isoform of BTK (labeled as BTKi). Both RAJI and Hek293T cells express SYK and IK1. [A2] Upper panel: BTK Western blot analysis of whole cell lysates from Hek293T cells treated with medium only (CON), BTK siRNA or Ku80 siRNA that was used as a control. Each siRNA was used at a 50 nM concentration. BTK siRNA (but not Ku80 siRNA) resulted in depletion of BTK protein without a decrease in the amount of IK protein. Lower panel: Ku80 Western blot analysis of whole cell lysates from Hek293T cells treated with medium only (CON), BTK siRNA or Ku80 siRNA. BTK siRNA did not affect the expression level of the control protein Ku80. In contrast, Ku80 siRNA resulted in depletion of Ku80 protein. [A3] Additional Controls. SYK vs. IK Western blot analysis of whole cell lysates from 293T cells treated with medium only (CON), SYK siRNA or BTK siRNA [13]. BTK siRNA did not cause a decrease in the amount of SYK or IK proteins [modified from Figure 2 of our recent open access article published in PNAS [13]. [A4] EMSAs were performed on nuclear extracts (NE) from untreated control (CON) Hek293T cells as well as Hek293T cells treated for 72 h with BTK siRNA (50 nM) using biotin-labeled DNA probe IK-BS1. Both 0.4 µg (1×) and 4 µg (10×) amounts of NE were used. IK activity was measured by the electrophoretic mobility shifts of the biotin-labeled IK-BS1 probe, representing IK-containing nuclear complexes (indicated with arrow heads). The position of the probe is also indicated with an arrowhead at the bottom of the gel. The biotin-labeled DNA was detected using a streptavidin-horseradish peroxidase conjugate and a chemiluminescent substrate. The membrane was exposed to X-ray film and developed with a film processor. [B] IK binding sites of validated IK target genes. By cross-referencing IK-regulated gene set (GSE323211) with the archived CHiPseq data (GSM803110) the location of potential IK binding sites for validated IK target genes Itga4 (NM_010576; chr2:79095583–79173271), Eif4e3 (NM_025829; chr6:99575131–99616765), Kif23 (NM_024245; chr9:61765085–61794606) and Tnfaip8l2 (NM_027206; chr3:94943443–94946282) was visualized in the mouse mm9 reference database using the Integrative Genomics Browser. [C] RT-PCR was used to examine the expression levels of randomly selected IK target genes after 72 h treatment with medium alone (CON), 50 nM scrambled siRNA (sc-siRNA), IKZF1 siRNA vs. BTK siRNA. C1: Expression levels of 4 randomly selected IK target genes were reduced by siRNA-mediated depletion of BTK, whereas treatment with sc-siRNA had no such effect. C2: Included as a positive control, IKZF1 siRNA also abrogated or reduced the expression of all 4 IK target genes.
Figure 4.
Phosphorylation and Activation of Recombinant Ikaros by Recombinant BTK and BTK-KD.
[A] Recombinant full-length BTK as well as BTK-KD showed autophosphorylation and it also phosphorylated MBP-tagged recombinant IK1 in hot kinase assays in the presence of [gamma-32P]ATP. Depicted is the autoradiograph of the kinase reaction products. The positions of the autophosphorylated BTK kinase band and BTK kinase domain band as well as the phosphorylated recombinant IK band are indicated with arrows. [B] Phospho amino acid analysis of the BTK phosphorylated MBP-IK1 band showed phosphorylation on tyrosine and serine. The 32P-labeled MBP-IK1 band in [A] was isolated and subjected to PAA. The positions of ninhydrin-stained phosphoamino acid standards (phosphoserine [S], phosphothreonine [T], and phosphotyrosine [Y]) are indicated with circles. [C] EMSA's were performed with a 32P-labeled double-stranded IK-BS1 oligonucleotide probe containing a high-affinity IK binding site (1 ng/sample, 100,000 cpm) and purified MBP-tagged recombinant IK1 protein (200 ng/sample) that has or has not been phosphorylated by recombinant BTK as in panel A. Lane 1, IK-BS1 probe only. Lane 2, unphosphorylated IK1 (200 ng) was mixed with 1 ng radiolabeled IK-BS1. Lane 3/Lane 4, BTK-phosphorylated MBP-IK1 (MBP-IK1Phos(BTK)) was mixed after a 5 min vs. 30 min kinase assay with 1 ng radiolabeled IK-BS1. MBP-IK1Phos(BTK) exhibited augmented binding to IK-BS1 when compared to MBP-IK1. [D&E] Mass spectrographs of phosphopeptides from BTK-phosphorylated recombinant IK1 protein. A MALDI-TOF/TOF mass spectrometry analysis was performed on trypsin-digested recombinant IK1 after an in vitro kinase reaction with purified recombinant BTK. Supel-Tips (Sigma-Aldrich) were used for phosphopeptide enrichment. Tryptic peptides were desalted and concentrated using the Millipore C18 reverse phase Zip-Tips column, eluted in 0.5 μL of matrix solution and spotted on the MALDI plate. MALDI-TOF MS was performed on an AB Sciex Proteomics Analyzer. MS spectra were acquired in reflection positive ion mode, averaging 4000 laser shots/per spectrum. The masses of the peptides after neutral loss 1 (−98 kDa) or 2 (−196 kDa) are indicated with arrows. [D] CID spectrum shows neutral loss of 1 phosphate indicating that S168 serves as a target phosphorylation site for BTK. [E] CID spectrum reveals neutral loss of 2 phosphates indicating that S214 and S215 residues of IK serve as unique target phosphorylation sites for BTK.
Figure 5.
BTK phosphorylation sites of Ikaros.
[A] We identified Ser214 (S214) and Ser215 (S215) as two unique BTK phosphorylation sites within the mouse IK peptide 214SSLEEHK220 corresponding to a consensus sequence encoded by Exon 5 and found in IK from mouse (NM_001025597, human (NM_006060.4), and chicken (NM_205088). [B] Alignment of mouse, human, and chicken IK protein segments containing the identified BTK phosphorylation sites. [C] Schematic diagram of BTK-IK (203–224) complex. BTK is shown as a molecular surface colored in gray, and IK is shown as secondary structure colored in green. Key residues in catalytic site of BTK, ATP and substrate residues (Ser214 and Ser215) in the peptide of IK are shown as a stick model. The model was built by Modeler [1] and shown with Pymol. [D] Loss of IK DNA binding activity by BTK-resistance mutations. EMSAs were performed on nuclear extracts (NE) from Hek293T cells transfected with expression vectors for wildtype (WT) or mutant IK proteins using the Thermo Scientific LightShift Chemiluminescent EMSA Kit and biotin-labeled DNA probe gamma-satellite A. IK activity was measured by the electrophoretic mobility shifts of the biotin-labeled probe, representing IK-containing nuclear complexes (indicated with arrow head). The biotin-labeled DNA was detected using streptavidin-horseradish peroxidase conjugate and a chemiluminescent substrate. The membrane was exposed to X-ray film and developed with a film processor. [E] Confocal two-color merge image of a representative Hek293T cell expressing FLAG-tagged wildtype IK protein (green) in the DAPI-stained (blue) nucleus. (System Magnification: 630×). [F] Confocal two-color merge image of representative Hek293T cells expressing FLAG-tagged mutant IK protein with alanine substitutions at BTK phosphorylation sites S214 and S215 (green) in the cytoplasm outside the DAPI-stained nucleus. (System Magnification: 630×). To detect the FLAG-tagged wildtype (in E) and BTK-phosphorylation site mutant IK proteins (in F), cells were stained with a monoclonal mouse anti-DDK antibody and a secondary goat anti-mouse antibody conjugated with Alexa Fluor 488. Fluorescent cells were imaged using the PerkinElmer Ultraviewer Confocal Dual Spinning Disc Scanner (Shelton, CT).