As mentioned above, MAPKs typically form multi-tiered pathways, receiving input several levels above the actual MAP kinase. In contrast to the relatively simple, phosphorylation-dependent activation mechanism of MAPKs and MAP2Ks, MAP3Ks have stunningly complex regulation. Many of the better-known MAP3Ks, such as c-Raf, MEKK4 or MLK3 require multiple steps for their activation. These are typically allosterically-controlled enzymes, tightly locked into an inactive state by multiple mechanisms. The first step en route to their activation consists of relieving their autoinhibition by a smaller ligand (such as Ras for c-Raf, GADD45 for MEKK4 or Cdc42 for MLK3). This commonly (but not always) happens at the cell membrane, where most of their activators are bound (note that small G-proteins are constitutively membrane-associated due to prenylation). That step is followed by side-to-side homo- and heterodimerisation of their now accessible kinase domains. Recently determined complex structures reveal that the dimers are formed in an orientation that leaves both their substrate-binding regions free. Importantly, this dimerisation event also forces the MAP3 kinase domains to adopt a partially active conformation. Full activity is only achieved once these dimers transphosphorylate each other on their activation loops. The latter step can also be achieved or aided by auxiliary protein kinases (MAP4 kinases, members of the Ste20 family). Once a MAP3 kinase is fully active, it may phosphorylate its substrate MAP2 kinases, which in turn will phosphorylate their MAP kinase substrates.