The SRS contains a binding site for the Ubl-conjugating (E2) enzyme mUBC9. (A) Alignment of amino acid sequences of UBC9 from different species. mUBC9 was cloned by the yeast two-hybrid screen using HIPK2 as bait, and the deduced amino acid sequence was aligned with known UBC9s from different species. Percent identity is indicated within the parentheses. (B) In vitro interaction of mUBC9 with HIPK2. In vitro pull-down assays were performed with a GST-mUBC9 fusion protein (lane 3) and in vitro-translated ³⁵S-labeled HIPK2. Input sample (lane 1) contained 20% of the amount used for binding. GST (lane 2), pull-down assay with glutathione-Sepharose-bound GST protein as control. Bound HIPK2 is indicated by arrowhead. (C) Deletion analysis of the HIPK2 interaction domain with mUBC9. Various baits (D1–D10) were constructed, and the interactions with mUBC9 were assayed in yeast cells and are shown as β-galactosidase activity. The coding region of the HIPK2 fragment used is indicated by amino acid numbers. The mUBC9 binding domain is indicated by a double-headed arrow below D10. (D) The mUBC9 binding domain is conserved among different HIPKs. The amino acid sequence of the mUBC9 binding domain of HIPK2 (indicated by bracket) is aligned with the corresponding regions of HIPK1 and HIPK3. Black highlighting with white characters indicates identical amino acids, and light gray-highlighted amino acids represent conservative amino acid replacements. Numbers indicate amino acid residues.

Localization of SUMO-1-modified HIPK2 to nuclear speckles (dots). (A) Fractionation of SUMO-1-modified HIPK2. Cellular extracts from CV-1 cells cotransfected with GFP-SUMO-1 and Myc-HIPK2 expression vectors were fractionated. Proteins from each fraction were separated on SDS/polyacrylamide gradient (4–12%) gels and analyzed by Western blotting using a mouse anti-Myc Ab. HIPK2 and SUMO-1-modified HIPK2 are detected in the nuclear fraction (lane 3) and in the Nonidet P-40-insoluble fractions (lanes 7 and 8). SUMO-1-modified HIPK2 and unmodified HIPK2 are marked by bracket and arrowhead, respectively. (B) Confocal microscopic image of the nucleus showing colocalization of HIPK2 and SUMO-1 to nuclear speckles (dots). CV-1 cells overexpressing Myc-HIPK2 and GFP-SUMO-1 were fixed and subjected to immunofluorescence staining with a mouse anti-Myc mAb. The red signal (HIPK2) is obtained with an anti-mouse IgG rhodamine red-conjugated secondary Ab, whereas the green signal (SUMO-1) is obtained with GFP fluorescence. Superimposing two colors (MERGE) results in a yellow signal, indicating colocalization of the two proteins.

HIPK2 contains multiple NLSs and localizes to nuclear speckles (dots) by means of a SRS. (A) Subcellular localization of various GFP-HIPK2 fusion proteins. CV-1 cells were transfected with various GFP-HIPK2 fusion constructs, and GFP fluorescence was visualized 48 hr after transfection. Constructs A–L correspond to the construct in B used for transfection. (B) Schematics of various GFP-HIPK2 constructs (constructs A–L). Locations of NLSs and SRS are shown under the schematic of the HIPK2 protein. HIPK2 contains three NLSs (NLS1–3, arrowheads) and a SRS (amino acids 860–967) that overlaps with the PEST sequence and NLS3. The coding region that was fused to the GFP ORF is indicated by amino acid numbers in each construct. KD, protein kinase domain; ID, interaction domain with homeodomain proteins; PEST, PEST sequence; YH, a C-terminal region enriched in tyrosine and histidine residues; S, speckles; ++, strongly positive; +, positive both in the cytoplasm (C) and the nucleus (N); +/−, weakly positive; −, negative.

Covalent modification of HIPK2 by SUMO-1. (A) Nuclear extracts from C2C12 cells were subjected to immunoprecipitation (IP) with an anti-SUMO-1 Ab (lane 2) or control IgG (lane 3) followed by Western blot analysis with an anti-HIPK2 Ab. Small bracket indicates broad bands that may include both unmodified and modified HIPK2, and the arrowhead indicates possible SUMO-1-modified HIPK2 band (SUMO-HIPK2). h.c., Ig heavy chain. (B) CV-1 cells were transfected with empty vector (lanes 1, 5, and 9) or the indicated expression vector containing a cDNA insert encoding Myc-HIPK2 (lanes 2, 6, and 10), GFP-SUMO-1 (lanes 3, 7, and 11), or both Myc-HIPK2 and GFP-SUMO-1 (lanes 4, 8, and 12). Cell lysates were analyzed by SDS/PAGE, followed by Western blotting with a mouse anti-GFP mAb (lanes 1–4) or with a mouse anti-Myc mAb (lanes 5–8). The SUMO-1-modified proteins were immunoprecipitated from the cell lysate (lanes 9–12) with a rabbit anti-GFP polyclonal Ab and analyzed by Western blotting with a mouse anti-Myc mAb. SUMO-1-modified proteins are marked by a large bracket (lanes 3 and 4), and the SUMO-1-modified HIPK2 is marked by a small bracket (lanes 8 and 12). Arrowhead indicates unmodified HIPK2 (lanes 6 and 8), and open arrowhead indicates the GFP-SUMO-1 monomer band (lanes 3 and 4). Molecular size markers are shown on the left in kDa.

Correlation of SUMO-1 modification of HIPK2 with the compartmentalization of HIPK2 to nuclear speckles (dots) within the nucleus. (A) GFP-HIPK2 constructs were transfected into CV-1 cells, and GFP fluorescence was visualized 48 hr after transfection (Lower). Schematics of GFP-HIPK2 constructs are shown (Upper). (B) Covalent modification of HIPK2 by SUMO-1. Different Myc–HIPK2 constructs were transfected into CV-1 cells in the presence (+) of GFP-SUMO-1, and SUMO-1-modified HIPK2 was detected by immunoprecipitation with an anti-GFP Ab (lanes 3, 6, and 9) followed by Western blot analysis with an anti-Myc Ab.