Apoptosis signal-regulating kinase (ASK1) is a mitogen-activated protein kinase (MAPK) that transduces apoptotic signals from a variety of stresses. We have shown previously that alpha subunits of heterotrimeric G12 and G13 proteins stimulate ASK1 kinase activity and ASK1-dependent apoptosis (1). Here, we report a novel mechanism of G-protein-dependent regulation of ASK1. We demonstrated that G13 forms a complex with ASK1 in an activation-independent manner. Both N- and C-terminal regulatory domains of ASK1 were essential for the efficient interaction, while its kinase domain was not required. Formation of the G13-ASK1 complex was enhanced by JNK-interacting leucine zipper protein, JLP. Constitutively activated G13Q226L increased ASK1 expression. Short-term activation of a serotonin 5-HT₄ receptor that is coupled to G13 also increased ASK1 expression. Importantly, prolonged activation of 5-HT₄ receptor in COS-7 cells or prolonged treatment of human umbilical vein endothelial cells with thrombin concomitantly down-regulated both G13 and ASK1. Data showed that G13Q226L reduced the rate of ASK1 degradation, decreased ASK1 ubiquitination, and reduced association of ASK1 with an E3 ubiquitin ligase CHIP, previously shown to mediate ASK1 degradation. Our findings indicate that ASK1 expression levels can be regulated by G13, at least in part via control of ASK1 ubiquitination and degradation.--Kutuzov, M. A., Andreeva, A. V., Voyno-Yasenetskaya, T. A. Regulation of apoptosis signal-regulating kinase 1 degradation by G13.