MAP kinases phosphorylate specific groups of substrate proteins. Here we show that the amino acid sequence FXFP is an evolutionarily conserved docking site that mediates ERK MAP kinase binding to substrates in multiple protein families. FXFP and the D box, a different docking site, form a modular recognition system, as they can function independently or in combination. FXFP is specific for ERK, whereas the D box mediates binding to ERK and JNK MAP kinase, suggesting that the partially overlapping substrate specificities of ERK and JNK result from recognition of shared and unique docking sites. These findings enabled us to predict new ERK substrates and design peptide inhibitors of ERK that functioned in vitro and in vivo. Mitogen-activated protein (MAP) kinases are components of signaling cascades that regulate normal development and pathological processes such as oncogenesis. MAP kinases were identified during biochemical searches for serine/threonine-specific protein kinases stimulated by growth factors in vertebrate cells (for review, see Sturgill and Wu 1991). MAP kinases were also identified in screens for mutations that affect intercellular signaling in yeast, worms, and flies (for review, see Ferrell 1996). Together, these investigations revealed that MAP kinases function in many cell types, are regulated by a diverse group of extracellular stimuli, and mediate a wide variety of cellular responses. MAP kinases can be divided into subfamilies based on specific conserved residues, particularly a TXY motif in the activation loop (Ferrell 1996). The three best-characterized subfamilies in vertebrates are named extracellular-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK, also called stress-activated protein kinase), and p38. There are probably several additional vertebrate MAP kinase subfamilies, since Saccharomyces cerevisiae contains six different MAP kinases (Madhani and Fink 1998). Here we use the name MAP kinase to refer to all members of the family, and the names ERK, JNK, and p38 to refer to members of those subfamilies.MAP kinases function in modules composed of three protein kinases (for review, see Marshall 1994). MAP kinase kinase kinases, such as Raf-1, phosphorylate and thereby activate MAP kinase kinases, such as MEK (MAP kinase kinase or ERK kinase). MAP kinase kinases are serine/threonine and tyrosine-specific protein kinases that phosphorylate the TXY motif and thereby activate MAP kinases. In general, MAP kinases in different subfamilies are members of separate modules and are regulated by distinct extracellular stimuli (for review, see Whitmarsh and Davis 1996). For example, ERK is activated strongly by receptor tyrosine kinases (RTK) such as the epidermal growth factor receptor, whereas JNK is activated strongly by stress stimuli such as ultraviolet light. Several of the signaling pathways leading from extracellular stimuli to the activation of a MAP kinase module are well defined, whereas others have yet to be characterized in detail. Whereas the upstream signaling events that r...