Raf-1 plays important roles in cell proliferation, differentiation, and survival. However, the unique and essential function of Raf-1 is anti-apoptotic. The molecules that mediate Raf-1's anti-apoptotic function are not known. In the course of identifying new substrates of Raf-1, we found that the Raf-1 kinase domain interacted with apoptosis-linked gene-2 (ALG-2) in yeast two-hybrid system. Our further studies showed that Raf-1 phosphorylated ALG-2 in an in vitro kinase assay. We also found that apoptosis signal-regulating kinase 1 (ASK1) strongly phosphorylated ALG-2. Importantly, Raf-1 blocks the ASK1-dependent ALG-2 phosphorylation. Since ALG-2 associates with ASK1, and both ASK1 and ALG-2 are involved in apoptosis, our observations indicate that Raf-1 may mediate its anti-apoptotic function by interrupting ASK1-dependent phosphorylation of ALG-2. (c) 2005 Elsevier Inc. All rights reserved.