Novel mixed-lineage kinase protein zipper sterile-a-motif kinase (ZAK) was first cloned by our laboratory. Lung cancer is the leading cause of cancer-related death in the world, including in Taiwan. Here, we wanted to investigate whether ZAK plays a potential role in lung cancer development. First, Western blot analysis results demonstrated that four cell lines expressed high levels of ZAK from among a panel of 10 lung cancer cell lines, and two of three normal lung cells expressed ZAK. ZAK gene expressions were down-regulated in lung cancers by real-time PCR analysis. Overexpression of ZAK suppressed cell proliferation in parallel with increased phosphorylated levels of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). In contrast, ZAK silencing cells inhibited the expressions of phosphorylated ERK and JNK without affecting the expression of phosphorylated p38. The effect of the decreased cell growth rate was significantly but incompletely reversed when ZAK-overexpressing cells were treated with a specific ERK or JNK inhibitor. Moreover, c-Fos and c-Jun, the major downstream components of MAPKs, were up-regulated by ERK and JNK, respectively. When ZAK-overexpressing cells introduced with c-Jun RNA interference (RNAi), the activator protein-1 (AP-1) transcription activity detected by a secreted alkaline phosphatase (SEAP) assay was suppressed and the decreased cell number was reversed compared with the control RNAi-treated group. More importantly, ZAK significantly depressed tumor growth in in vivo study. Taken together, results from both in vitro and in vivo studies indicated that the decrease of lung cancer cell proliferation by ZAK may involve the ERK and JNK pathways via an AP-1 transcription factor. (Cancer Sci 2010; 101: 1374-1381 T he mixed-lineage kinases (MLKs) are a family of serine ⁄ threonine protein kinases that function as mitogen-activated protein kinase (MAPK) kinase kinases (MAPKKKs).(1)The MLKs cluster into three subgroups based on the domain arrangements and sequence similarity within their catalytic domains: the MLKs, the dual-leucine-zipper-bearing kinases, and zipper sterile-a-motif kinase (ZAK). We are the first group to clone ZAK in 2000(2) (GenBank accession number: AF238255). This cDNA has 2456 bp and encodes a protein of 800 amino acids that contains a kinase catalytic domain, a leucine zipper, and a sterile-a-motif (SAM). Our ZAK is also known as ZAK-a or MLK-like MAP triple kinase-a (MLTK-a).(1,3) ZAK-b is an alternative splicing product and is also referred to as MLTK-b or MLK7. ZAK-b is identical to ZAK-a from the amino terminus to the zipper domain, but then diverges and terminates shortly thereafter, so it lacks a SAM domain. (1,3) Similar to other MLK family members, the MLTKs regulate signaling of the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase. The transcription factor activator protein-1 (AP-1) regulates a wide range of cellular processes, including cell proliferation, apoptosis, survival, and differe...