Prostate apoptosis response-4 (Par-4) was initially identified as a gene product up-regulated in prostate cancer cells undergoing apoptosis. In rat fibroblasts, coexpression of Par-4 and its interaction partner DAP-like kinase (Dlk, which is also known as zipper-interacting protein kinase [ZIPK]) induces relocation of the kinase from the nucleus to the actin filament system, followed by extensive myosin light chain (MLC) phosphorylation and induction of apoptosis. Our analyses show that the synergistic proapoptotic effect of Dlk/Par-4 complexes is abrogated when either Dlk/Par-4 interaction or Dlk kinase activity is impaired. In vitro phosphorylation assays employing Dlk and Par-4 phosphorylation mutants carrying alanine substitutions for residues S154, T155, S220, or S249, respectively, identified T155 as the major Par-4 phosphorylation site of Dlk. Coexpression experiments in REF52.2 cells revealed that phosphorylation of Par-4 at T155 by Dlk was essential for apoptosis induction in vivo. In the presence of the Par-4 T155A mutant Dlk was partially recruited to actin filaments but resided mainly in the nucleus. Consequently, apoptosis was not induced in Dlk/Par-4 T155A-expressing cells. In vivo phosphorylation of Par-4 at T155 was demonstrated with a phospho-specific Par-4 antibody. Our results demonstrate that Dlk-mediated phosphorylation of Par-4 at T155 is a crucial event in Dlk/Par-4-induced apoptosis.