Furthermore, the rescuing activity of DLK-1L was strongly attenuated by co-overexpression with DLK-1S ( Figures 1C and 1D, juEx2802, juEx2813). This inhibitory effect of DLK-1S was eliminated when the LZ domain was deleted from DLK-1S ( Figure S2C). However, expression of a kinase-dead mutant DLK-1S(K162A), in which the Lys162 at the ATP binding

site of the kinase domain was mutated to Ala ( Nakata et al., 2005), inhibited DLK-1L to a similar degree as did wild-type DLK-1S ( Figure S2C). These data suggest that the ability of DLK-1S to inhibit DLK-1L requires its LZ domain but not its kinase activity. As a further test for the role of DLK-1S, we expressed various DLK-1 constructs in the wild-type background ( Figure S2D). Overexpression Obeticholic Acid research buy of DLK-1L alone caused abnormal neuronal development, whereas overexpression of DLK-1(mini) gene had a much weaker effect. Removing intron 7 from DLK-1(mini), which would prevent production of DLK-1S, resulted in gain-of-function effects similar to DLK-1(L). Finally, to address whether transgenically expressed DLK-1S could interfere

with endogenous DLK-1L, we overexpressed DLK-1S in rpm-1(lf) single mutants and observed this website significant suppression of rpm-1(lf) phenotypes ( Figure S3A). Together, these analyses demonstrate that despite sharing identical kinase and LZ domains, DLK-1S is a potent inhibitor of DLK-1L function. If DLK-1S acts as an endogenous inhibitory isoform, how does DLK-1L become activated at all? Since DLK-1L and DLK-1S differ only in their C termini, we hypothesized that the C terminus of DLK-1L may contain elements important

for its kinase activation and that DLK-1S may act by preventing the interactions between the such elements and the kinase domain. To test this idea, we generated a series of DLK-1L variants in which the C terminus was either truncated or contained internal deletions (Supplemental Experimental Procedures) and assayed rescuing activity of these constructs in the dlk-1(lf); rpm-1(lf) double mutant strain ( Figure 2, Table S2). We found that a region of 25 amino acids from residues 856 to 881 in the DLK-1L C terminus was necessary for DLK-1L activity ( Figure 2, juEx3586). Remarkably, a construct lacking all of the DLK-1L C terminus except for aa 856–881 recapitulated the activity of the full-length DLK-1L ( Figure 2, juEx3657), suggesting that this region is sufficient for DLK-1L regulation. Upon closer inspection of the amino acid sequences, we found a six residue motif SDGLSD (aa 874–879, hereafter referred to as the hexapeptide) that is completely conserved between C. elegans DLK-1 and vertebrate MAP3K13/LZK ( Figure 3A); the remainder of the C termini of these kinases show little sequence conservation. Moreover, dlk-1(ju620), a strong loss-of-function mutation, results in a missense alteration (G870E) adjacent to this hexapeptide.