Fig. 3.

HIPK2 directly phosphorylates a novel ATF1 site, not Ser63. (A) Increasing amounts of recombinant HIPK2 (amino acids 165-564) was mixed with 2 μg of bacterially expressed His-ATF1 or His-ATF1 Ser63Ala mutant and incubated in the presence of γ-³²P-ATP at 30°C for 20 minutes. Samples were separated on SDS-PAGE and visualized by autoradiography. PKA was used as a control. Asterisk indicates autophosphorylated HIPK2. Coomassie Blue staining of the same gel is shown (bottom). The stained band below the 76 kDa marker is BSA added to recombinant HIPK2 solution as a carrier protein. The molecular weight of the recombinant HIPK2 (a.a.165–564) is approximately 55 kDa. (B) Wt HIPK2 or kd HIPK2 (K221R) plasmid DNA were co-expressed with wt ATF1 or Ser63Ala-mutant ATF1 in HEK293 cells. Twenty-four hours after transfection, total cell lysates were collected and 40 μg of samples were separated on SDS-PAGE and then analyzed by western blotting using anti-phospho ATF1 Ser63 antibody (top), anti-ATF1 antibody (middle) or anti-HIPK2 antibody (bottom). Cell lysate with co-expression of PKA and ATF1 was used as a positive control of ATF1 Ser63 phosphorylation (lane 8).