Journal of Cellular Biochemistry 101:34–43 (2007) The MEK Kinases MEKK4/Ssk2p Facilitate Complexity in the Stress Signaling Responses of Diverse Systems Blaine T. Bettinger and David C. Amberg* Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 E. Adams St., Syracuse, New York 13210 Abstract The mammalian JNK/p38 MAP kinase kinase kinase MEKK4 and the Saccharomyces cerevisiae Ssk2p are highly homologous. MEKK4 can replace all of the known functions of Ssk2p in yeast, including functioning in the high osmolarity glycerol (HOG) MAPK pathway and the recently described actin recovery pathway. MEKK4 and Ssk2p share a number of conserved domains and appear to be activated by a similar mechanism. Binding of an activating protein to the N-terminal region alleviates auto-inhibition and causes the kinase to auto-phosphorylate, resulting in activation. In this review we will examine the role of the MAP kinase kinase kinase isoform Ssk2p/MEKK4 in the adaptation of both yeast and mammalian systems to specific external stimuli. Recent work has provided a wealth of information about the activation, regulation, and functions of these MEKK kinases to extra-cellular signals. We will also highlight evidence supporting a role for MEKK4 in mediating actin recovery following osmotic shock in mammalian cells. J. Cell. Biochem. 101: 34–43, 2007. ß 2007 Wiley-Liss, Inc. Key words: SSK2; MEKK4; MTK1; osmotic stress; MAP kinase pathway; actin Eukaryotic stress-activated mitogen-activated protein kinase (SAPK) pathways have evolved to respond to a multitude of extra-cellular stresses, such as physical (UV radiation, osmo- tic variability, heat), chemical (pH, oxidative stress), and biological (tissue or organ stressors, DNA damage, cytokines, pheromones) stresses [Kyriakis and Avruch, 2001]. SAPK pathways, which have been highly conserved throughout evolution, relay signals through a sequential cascade of proteins that begins with the activa- tion of a MEK kinase, also called a MAP kinase kinase kinase (MAPKKK). The MAPKKK phos- phorylates and activates an intermediate MAP kinase kinase (MKK) (MKK3, MKK4, MKK6, and/or MKK7, for example), which in turn phosphorylates and activates a MAP kinase (such as JNK or p38). Activated MAP kinases elicit the appropriate cellular response through the phosphorylation of numerous proteins including transcription factors. This simple architecture has been recycled throughout nature to build a myriad of parallel pathways that fine tune the cellular responses to diverse stress stimuli. Surprisingly, parallel SAPK pathways use many of the same signaling intermediates. The JNK and p38 cascades, for example, share the MAPKKK MEKK2, 3, and 4 as well as the intermediate MKK4 [Kyriakis and Avruch, 2001]. To establish and preserve specificity of signaling, nature has evolved a number of regulatory mechanisms including compartmen- talization (scaffolding and localization), the use of activating or inhibitory protein interactions, and tissue-specific expression of pathway com- ponents. Recent work has significantly advanc- ed our understanding of another mechanism of specificity, the use of multiple MAPKKK iso- forms in a single pathway [Schlesinger et al., 1998; Hagemann and Blank, 2001; Uhlik et al., 2004]. In this review we will examine the role of the MAPKKKs Ssk2p/MEKK4 in the adapta- tion of both yeast and mammalian systems to specific external stimuli. The mammalian p38 pathway employs both MEKK3 and MEKK4 to phosphorylate and activate MKK6 [Deacon and ß 2007 Wiley-Liss, Inc. Grant sponsor: National Institutes of Health; Grant number: GM-56189. *Correspondence to: David C. Amberg, Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 E. Adams St., Syracuse, NY 13210. E-mail: ambergd@upstate.edu Received 17 November 2006; Accepted 9 January 2007 DOI 10.1002/jcb.21289