FIGURE 1.

c-Abl phosphorylates HIPK2 at several sites. A, schematic representation of HIPK2 and truncation mutants. HIPK2 autophosphorylation site is indicated in bold. Sequence in the region of amino acids 350–363 is shown. The five putative c-Abl consensus phosphorylation sites are indicated, as well as HIPK2 kinase domain and Siah-1 interaction domain. All constructs have a FLAG tag at the N terminus. B, Hep3B cells were treated with 10 μm STI571 1 h prior to IR at 20 Gy. Cells were harvested 2 days after IR, and anti-HIPK2 was used for immunoprecipitation. Immunoprecipitated and total samples were analyzed by SDS-PAGE and immunoblotting. Phospho-Tyr (PY20) detects phosphotyrosine. The anti-HIPK2 C1 antibody was used for IP, and anti-HIPK2 rb1 was used for immunoblot detection. C, HEK293 cells were transfected with pCDNA3 FLAG-HIPK2 wild type and K221A and with constitutively active c-Abl Δ1–81 or with pCDNA3 empty vector, as indicated. Cells were harvested 24 h after transfection, and anti-FLAG was used for immunoprecipitation. Cells were analyzed as in B. D, HEK293 cells were transfected with pCDNA3 FLAG-HIPK2 wild type and truncation mutants and with c-Abl Δ1–81, as indicated. Cells were analyzed as in B. E, HEK293 cells were transfected with pCDNA3 FLAG-HIPK2 wild type, 1–1019 and 1–1019 Y354F mutants, and c-Abl Δ1–81, as indicated. Cells were analyzed as in B. F, HEK293 cells were transfected with pCDNA3 FLAG-HIPK2 1–1019 wild type and point mutants and with active c-Abl, as indicated. Cells were analyzed as in B. G, HEK293 cells were transfected with pEFIRES FLAG-HIPK2 and active c-Abl constructs, as indicated, and cells were analyzed as in B.