Multiple docking sites on substrateproteins form a modular systemthat mediates recognition by ERKMAP kinaseDave Jacobs,1Danielle Glossip,1Heming Xing,2,3Anthony J. Muslin,2,3and Kerry Kornfeld1,41Department of Molecular Biology and Pharmacology,2Department of Medicine,3Department of Cell Biologyand Physiology, Washington University School of Medicine, St. Louis, Missouri 63110 USAMAP kinases phosphorylate specific groups of substrate proteins. Here we show that the amino acid sequenceFXFP is an evolutionarily conserved docking site that mediates ERK MAP kinase binding to substrates inmultiple protein families. FXFP and the D box, a different docking site, form a modular recognition system, asthey can function independently or in combination. FXFP is specific for ERK, whereas the D box mediatesbinding to ERK and JNK MAP kinase, suggesting that the partially overlapping substrate specificities of ERKand JNK result from recognition of shared and unique docking sites. These findings enabled us to predict newERK substrates and design peptide inhibitors of ERK that functioned in vitro and in vivo.[Key Words: MAP kinase; ERK; JNK; KSR; ETS transcription factor]Received October 14, 1998; revised version accepted December 11, 1998.Mitogen-activated protein (MAP) kinases are compo-nents of signaling cascades that regulate normal devel-opment and pathological processes such as oncogenesis.MAP kinases were identified during biochemicalsearches for serine/threonine-specific protein kinasesstimulated by growth factors in vertebrate cells (for re-view, see Sturgill and Wu 1991). MAP kinases were alsoidentified in screens for mutations that affect intercellu-lar signaling in yeast, worms, and flies (for review, seeFerrell 1996). Together, these investigations revealedthat MAP kinases function in many cell types, are regu-lated by a diverse group of extracellular stimuli, and me-diate a wide variety of cellular responses. MAP kinasescan be divided into subfamilies based on specific con-served residues, particularly a TXY motif in the activa-tion loop (Ferrell 1996). The three best-characterizedsubfamilies in vertebrates are named extracellular-regu-lated kinase (ERK), c-Jun amino-terminal kinase (JNK,also called stress-activated protein kinase), and p38.There are probably several additional vertebrate MAPkinase subfamilies, since Saccharomyces cerevisiae con-tains six different MAP kinases (Madhani and Fink1998). Here we use the name MAP kinase to refer to allmembers of the family, and the names ERK, JNK, andp38 to refer to members of those subfamilies.MAP kinases function in modules composed of threeprotein kinases (for review, see Marshall 1994). MAP ki-nase kinase kinases, such as Raf-1, phosphorylate andthereby activate MAP kinase kinases, such as MEK(MAP kinase kinase or ERK kinase). MAP kinase kinasesare serine/threonine and tyrosine-specific protein ki-nases that phosphorylate the TXY motif and thereby ac-tivate MAP kinases. In general, MAP kinases in differentsubfamilies are members of separate modules and areregulated by distinct extracellular stimuli (for review,see Whitmarsh and Davis 1996). For example, ERK isactivated strongly by receptor tyrosine kinases (RTK)such as the epidermal growth factor receptor, whereasJNK is activated strongly by stress stimuli such as ultra-violet light. Several of the signaling pathways leadingfrom extracellular stimuli to the activation of a MAPkinase module are well defined, whereas others have yetto be characterized in detail.Whereas the upstream signaling events that regulateMAP kinases have been characterized extensively, con-siderably less is known about how MAP kinases regulatecell fates and contribute to the specificity of signalingpathways. Important questions that remain largely un-answered include: (1) How do MAP kinases recognizespecific proteins as substrates? (2) What proteins arephosphorylated by a particular MAP kinase in differentcell types and in different organisms? Answers to thesequestions will illuminate how the same MAP kinase me-diates different cell fates in different developmental con-texts and how MAP kinases from separate subfamiliesmediate different cellular responses.4Corresponding author.E-MAIL [email protected]; FAX (314) 362-7058.GENES & DEVELOPMENT 13:163–175 © 1999 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/99 $5.00; www.genesdev.org 163In the case of ERK, >50 different proteins have beenreported to be substrates (for reviews, see Davis 1993;Karin 1995; Treisman 1996; Whitmarsh and Davis 1996;Madhani and Fink 1998). These include signaling pro-teins likely to function upstream of ERK such as Son-of-sevenless (Sos) guanine nucleotide exchange factor andMEK; signaling proteins likely to function downstreamof ERK such the protein kinase pp90rsk; transcriptionfactors such as c-Fos, GATA-2, c-Myc, and ETS proteinsincluding Elk-1, LIN-1, and Aop/Yan; and proteins in-volved in a wide variety of other processes. These find-ings suggest that ERK plays a central role in signal propa-gation and feedback regulation. Furthermore, ERK is atransition point between signaling proteins and regula-tors of differentiation, suggesting it makes an importantcontribution to the specificity of RTK–Ras–ERK signal-ing pathways. Although a large number of ERK sub-strates have been identified, the understanding of ERKfunction remains fragmentary, as ERK probably phos-phorylates different substrates in different cell types andthe cellular context of most substrates has yet to be de-fined. In addition, many ERK substrates probably havenot been identified. Substrates of JNK have been charac-terized less extensively, but it is notable that they in-clude proteins that are also phosphorylated by ERK, suchas Elk-1, as well as unique substrates (for review, seeMinden and Karin 1997).Little is known about how ERK recognizes such a di-verse group of substrates. Although the structure of ERKwas determined using X-ray crystallography (Zhang et al.1994; Canagarajah et al. 1997), this approach has not re-vealed how ERK interacts with substrate proteins, be-cause the structure of ERK bound to a substrate has yetto be determined. Studies of residues in substrate pro-teins that are phosphorylated by ERK and assays of pep-tide substrates identified a serine or threonine followedby a proline (S/TP) as the minimal consensus sequencefor phosphorylation by ERK (for review, see Davis 1993;Songyang et al. 1996). In addition, a proline at