Summary Mitogen-activated protein kinase (MAPK) cascades are core pathways mediating cellular responses to a wide variety of extracellular and intrinsic cues. In the past several years, we have made progress in understanding how connections are made in MAPK signaling networks, and we have exploited this knowledge to identify new MAPK substrates and regulators. We plan to extend these studies along two major lines of research. In the first, we will continue to investigate a role for non-catalytic “docking” interactions in mediating specificity and signaling output by MAPKs. We have developed yeast-based screening platforms to define sequence motifs selectively interacting with a conserved docking groove in the MAPK catalytic domain. We have so far applied these methods to a set of MAPKs including representatives of three major subfamilies (ERK, p38 and JNK). We plan to investigate novel MAPK substrates uncovered through these screens, with a focus on crosstalk between JNK and small GTPase signaling pathways. We further propose to expand our studies to include the remaining understudied MAPK groups, for which few substrates are known. To understand how selectivity is enforced in MAPK signaling systems, we will perform structural studies of MAPKs in complex with key regulators interacting with the docking groove and other ill-defined interfaces. Our second line of inquiry follows upon our recent discovery of the protein phosphatase 6 (PP6) complex as a MEK phosphatase that negatively regulates oncogenic ERK signaling. Despite being conserved throughout eukaryotes and essential to life in mammals, we know very little about the basic architecture and regulation of the complex. We will investigate the basis for PP6 complex assembly, and we will define how interactions with the catalytic and associated regulatory subunits confer substrate specificity. Finally, we will explore a role for PP6 in mediating a negative feedback loop in the ERK signaling pathway. Collectively, our proposed studies over the next five years will provide important new insight into the function of core signaling pathways relevant to normal physiology and human disease.