MAPKKKs provide stimulus specificity in signal transduction cascades and must be maintained in inactive states under basal conditions (Craig et al., 2008; L’Allemain, 1994). Most MAPKKKs contain regulatory domains in addition to the kinase domain. A well-known

example is Raf MAPKKK (Chong et al., 2003), which is maintained in an inactive state by binding of an N-terminal regulatory region to its kinase domain (Pumiglia et al., 1995). Release of this autoinhibition involves several proteins that bind to various regions of Raf, culminating with kinase activation by homo- or heterodimerization with its activation partner KSR (Fantl et al., 1994; Freed et al., 1994; Xing et al., 1997). Similarly, the N terminus of MTK1/MEKK4 MAPKKK binds to and inhibits its kinase domain, Selleckchem LDN 193189 and stress signals activate GADD45 proteins that bind to the N terminus, causing dissociation of the kinase domain and activation of MTK1 through protein dimerization (Mita et al., 2002; Miyake et al., 2007; Takekawa and Saito, 1998). To our knowledge, there is no exact precedent among MAPKKKs for the mechanism of DLK-1 activation. Although many MAP kinases are RGFP966 in vivo known to self-activate through kinase dimerization (Mita et al., 2002; Miyake et al., 2007; Takekawa and Saito, 1998), DLK-1L/S heteromeric binding does not activate DLK-1L. Notably, presence of the C-terminal domain in the DLK-1L/S heteromeric state is not sufficient to trigger DLK-1L activation. We envision

one possible activation mode that may involve conformational changes of the homomeric kinase domain mediated by an intermolecular interaction between the kinase domain

and the C-terminal activation domain (Figure S6B). In neurons, regulation of kinase activity by Ca2+ is well known. However, most Ca2+-dependent kinases either bind Ca2+ directly or are regulated by Ca2+-binding proteins, as in the case of CamKII (Meador et al., 1993). Ca2+/calmodulin binding causes phosphorylation of the internal CamKII peptide and changes the holoenzyme conformation, leading out to kinase activation (Yang and Schulman, 1999; Chao et al., 2011). Although several DLK kinase-binding proteins have been reported (Fukuyama et al., 2000; Horiuchi et al., 2007; Ghosh et al., 2011; Whitmarsh et al., 1998), none have been associated with Ca2+. Our studies therefore provide new insights for a structural understanding of DLK kinases. The C. elegans and Drosophila DLK kinases are orthologous to two closely related vertebrate MAPKKKs, DLK/MUK/ZPK/MAP3K12 and LZK/MAP3K13 ( Holzman et al., 1994; Sakuma et al., 1997). MAP3K12 and MAP3K13 display >95% sequence identity in their kinase domain, but their C termini diverge significantly. It has been shown that the LZ domain of MAP3K13 can mediate dimerization but is not sufficient to activate MAP3K13 ( Ikeda et al., 2001). MAP3K12 and MAP3K13 are known to activate different downstream kinases in cultured cells ( Ikeda et al., 2001; Nihalani et al.