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. 2019 Jan 1;9(1):94–107.

Figure 1.

Figure 1

Characterization of HIPK2 mutants found in acute myeloid leukemia and myelodysplastic syndrome patients. A. Schematic of HIPK2 and position of HIPK2 mutations. KD, protein kinase domain; ID, interaction domain with homeodomain proteins; SRS, speckle retention sequence; YH, tyrosine- and histidine-rich domain. Sequence alignment of human HIPK2 and mouse HIPK2 shows conservation (hHIPK2 Arg869 and mHIPK2 Arg861; hHIPK2 Asn958 and mHIPK2 Asn951). B. GST-pull down assay showing physical interaction of HIPK2-(800-1049) with SUMO-1. In vitro translated Myc-HIPK2-(800-1049), or the R861W or N951I mutant was incubated with equal amounts of either GST protein or GST-SUMO-1. Bound proteins were eluted and resolved by 8% SDS-PAGE, followed by Western blotting using anti-Myc antibody. Affinity purified GST or GST-SUMO-1 used in this assay are shown in the lower panel. Input shows 10% of the in vitro translated Myc-HIPK2 used in the binding reaction. C. Co-immunoprecipitation of Myc-HIPK2-(800-1049) with SUMO-1. An expression plasmid coding for either Myc-HIPK2-(800-1049), the R861W or N951I mutant was transfected into HeLa cells, together with an HA-SUMO-1 expression plasmid. Transfected cells were lysed and immunoprecipitated with anti-HA antibody, followed by Western blotting using anti-Myc antibody. D. Mutation of HIPK2 at Arg861 or Asn951 abolishes SUMO-1 conjugation to HIPK2. An expression plasmid encoding either Myc-HIPK2-(800-1049), R861W or N951I was transfected into HeLa cells with or without the SUMO-1 expression plasmid. SUMO-1 conjugation to HIPK2 was determined by Western blotting using anti-Myc antibody. E. In vitro translated Myc-HIPK2-(800-1049), the R861W or N951I mutant was subjected to in vitro SUMOylation assays. Reactions were terminated by adding sample buffer, followed by Western blotting using anti-Myc antibody. F. U2OS cells were transfected with GFP-HIPK2, the R861W or N951I mutant in combination with nuclear HA-PML-I or HA-PML-IV. Cells were fixed and immunostained 24 hrs after transfection. Images were obtained with a confocal fluorescent microscope at an excitation wavelength of 488 nm and 543 nm, respectively. G. Co-immunoprecipitation of Myc-HIPK2-(800-1049) with SUMOylated PML-IV. Either Myc-HIPK2-(800-1049), the R861W or N951I mutant expression plasmid was transfected into HeLa cells with the HA-PML-IV expression plasmid in the presence or absence of the GFP-SUMO-1 expression plasmid. Total cell lysates were immunoprecipitated with anti-Myc antibody, followed by Western blotting using anti-HA antibody.