the negatively charged CD-region. Together they recognize the so-called MAPK docking or D-motifs (also called kinase interaction motif / KIM). D-motifs essentially consist of one or two positively charged amino acids, followed by alternating hydrophobic residues (mostly leucines), typically upstream of the phosphorylation site by 10–50 amino acids. Many of the known MAPK substrates contain such D-motifs that can not only bind to, but also provide specific recognition by certain MAPKs. D-motifs are not restricted to substrates: MAP2 kinases also contain such motifs on their N-termini that are absolutely required for MAP2K-MAPK interaction and MAPK activation. Similarly, both dual-specificity MAP kinase phosphatases and MAP-specific tyrosine phosphatases bind to MAP kinases through the same docking site. D-motifs can even be found in certain MAPK pathway regulators and scaffolds (e.g. in the mammalian JIP proteins). Other, less well characterised substrate-binding sites also exist. One such site (the DEF site) is formed by the activation loop (when in the active conformation) and the MAP kinase-specific insert below it. This site can accommodate peptides with an FxFP consensus sequence, typically downstream of the phosphorylation site. Note that the latter site can only be found in proteins that need to selectively recognize the active MAP kinases, thus they are almost exclusively found in substrates. Different motifs may cooperate with each other, as in the Elk family of transcription factors, that possess both a D-motif and an FxFP motif. The presence of an FxFP motif in the KSR1 scaffold protein also serves to make it an ERK1/2 substrate, providing a negative feedback mechanism to set the correct strength of ERK1/2 activation. Scaffold proteins Since the discovery of Ste5 in yeast, scientists were on the hunt to discover similar non-enzymatic scaffolding pathway elements in mammals. There are indeed a number of proteins involved in ERK signaling, that can bind to multiple elements of the pathway: MP1 binds both MKK1/2 and ERK1/2, KSR1 and KSR2 can bind B-Raf or c-Raf, MKK1/2 and ERK1/2. Analogous proteins were also discovered for the JNK pathway: the JIP1/JIP2 and the JIP3/JIP4 families of proteins were all shown to bind MLKs, MKK7 and any JNK kinase. Unfortunately, unlike the yeast Ste5, the mechanisms by which they regulate MAPK activation are considerably less understood. While Ste5 actually forms a ternary complex with Ste7 and Fus3 to promote phosphorylation of the latter, known mammalian scaffold proteins appear to work by very different mechanisms. For example,