cGMP-dependent protein kinase-I (cGK-I) induces apoptosis in various cancer cell lines. However, the signaling mechanisms involved remain unknown. Using protein microarray technology, we identified a novel cGK substrate, death-associated protein kinase 2 (DAPK2), which is a Ca2+/calmodulin-regulated serine/threonine kinase. cGK-I phosphorylated DAPK2 at Ser(299), Ser(362) and Ser(368). Interestingly, a phospho-mimic mutant, DAPK2 S299D, significantly enhanced its kinase activity in the absence of Ca2+/calmodulin, while a S367D/S368D mutant did not. Overexpression of DAPK2 S299D also resulted in a twofold increase in apoptosis of human breast cancer MCF-7 cells as compared with wild-type DAPK2. These results suggest that DAPK2 is one of the targets of cGK-I in apoptosis induction. (C) 2012 Elsevier Inc. All rights reserved.