Figure 2.

Beclin 1 is a new substrate of DAPK. (A) Flag-tagged DAPK (100 ng) was incubated with GST (900 ng) or GST–beclin-1 (750 ng) in the presence of Ca²⁺, calmodulin and [γ-³³P]ATP for 30 min or 60 min. Phosphorylated proteins were visualized by X-ray film exposure, and GST/GST–beclin-1 levels were visualized by Ponceau S staining. The autophosphorylation of DAPK indicates that its catalytic activity was intact in all samples. (B) Flag-tagged DAPK (60 ng) was incubated with Flag-tagged beclin 1 (250 ng), which was purified from HEK293T cells, and a kinase assay was performed for 60 min. Where indicated (+LiCl), beclin-1-bound beads were first washed stringently in 0.5 M LiCl and 0.5 M KCl. Phosphorylated proteins were visualized by X-ray film exposure, and the levels of beclin 1 were visualized by Western blot analysis using beclin 1 antibodies. DAPK, death-associated protein kinase; GST, glutathione S-transferase; HEK, human embryonic kidney.