We wondered whether this region could be involved in Ca2+-dependent modulation of DLK-1 isoform-specific interactions. We found that DLK-1L and the mutants DLK-1L(Δ856–881), DLK-1L(Δ874–879), and DLK-1L(S874A, S878A) bound to DLK-1S to a similar degree under normal culture condition (Figures 8C, 8D, and S5A), consistent with the yeast two-hybrid interactions. However, ionomycin treatment did not cause detectable binding partner changes of the mutants DLK-1L(Δ856–881), DLK-1L(Δ874–879), see more and DLK-1L(S874A, S878A). We also found that DLK-1L(S874E, S878E) showed strong binding to itself even without ionomycin

treatment (Figure S5A). These results support the idea that C terminus of DLK-1L is required for the dissociation of DLK-1L/S heteromeric complexes caused by increasing Ca2+ levels. To test whether Ca2+ played a regulatory role in vivo, we next analyzed GFP-DLK-1L dynamics in egl-19(ad695 gf) animals, which is a gain-of-function mutation in the Ca2+ channel ( Kerr et al., 2000; Lee et al.,

1997). Previous studies have shown that egl-19(gf) enhances Ca2+ influx in Autophagy Compound Library ic50 PLM neurons after axotomy ( Ghosh-Roy et al., 2010). We found that egl-19(gf) mutants also displayed significantly increased accumulation of GFP-DLK-1L at cut sites, compared to wild-type ( Figure 8E, juEx2529). In contrast, neither DLK-1S nor DLK-1L(Δ856–881) showed local changes upon immediate axonal injury in egl-19(gf) or wild-type ( Figure 8E, juEx2531, juEx4932).

These data suggest that a transient increase in Ca2+ levels, as caused by axonal injury or synaptic activity, can trigger the release of DLK-1L from inhibition by DLK-1S and that this dissociation may be influenced by the phosphorylation state of the C-terminal hexapeptide. The DLK kinases play key roles in synapse and axon development and axon regeneration (Chen et al., 2011; Collins et al., 2006; Hammarlund et al., 2009; Itoh et al., 2009; Lewcock et al., 2007; Nakata et al., 2005; Xiong et al., 2010; Yan et al., 2009; Shin et al., 2012). In particular, timely activation of DLK kinases is critical for early responses Olopatadine to axonal injury (Chen et al., 2011; Hammarlund et al., 2009). In this study, we have uncovered a regulatory mechanism that endows C. elegans DLK-1 kinase with the ability to be rapidly activated by axon injury. We find that the short isoform, DLK-1S, acts as an endogenous inhibitor of the active long isoform DLK-1L. Our data support a model in which the balance between the active DLK-1L homomeric complexes and inactive DLK-1L/S heteromeric complexes can be spatially and temporally regulated by the conserved hexapeptide in a stimulus- or Ca2+-dependent manner ( Figure S6). This regulation is mediated via a C-terminal hexapeptide that is highly conserved in the DLK-1 and MAP3K13 family. Our observation that human MAP3K13 can functionally complement C.