These data are consistent with the conclusion that binding of DLK-1S to DLK-1L keeps DLK-1L inactive. We further found that green fluorescent protein (GFP)-tagged C terminus (aa 566–928) of DLK-1L recapitulated full-length DLK-1L localization Anticancer Compound Library ( Figure 5C). However, phosphomimetic or

nonphosphorylatable mutations of the hexapeptide did not change DLK-1L localization ( Figure S4). We conclude that the C terminus of DLK-1L is not only required for DLK-1L activity but also necessary for its subcellular localization in neurons. How might the isoform-dependent interaction of DLK-1 be regulated in vivo? To address this question, we focused on the function of DLK-1 in adult neurons. In dlk-1(lf) adult animals, injured axons fail to regrow ( Hammarlund et al., 2009; Yan et al., 2009). Overexpression of DLK-1(mini) completely rescued the regeneration failure of injured PLM axons in dlk-1(tm4024) mutants ( Figures 6A and 6B, juEx3757). Overexpression of DLK-1L not only rescued the regeneration failure, but also greatly enhanced the overall extent of axon regrowth (juEx2789) ( Hammarlund et al., 2009; Yan et al., 2009).

Paralleling our observations in developing neurons, overexpression of DLK-1S did not rescue the regeneration failure of dlk-1(lf) mutants (juEx2791) and blocked the regrowth-enhancing effects of DLK-1L (juEx2815). The inhibitory activity of DLK-1S required its LZ domain (juEx2881). Z VAD FMK However, DLK-1L(EE) with C-terminal phosphomimetic mutations showed regrowth-enhancing effects when coexpressed with Carnitine dehydrogenase DLK-1S (juEx4694), suggesting that DLK-1S is less able to inhibit DLK-1L, whose hexapeptide is phosphorylated. As axon regeneration is highly sensitive to the dosage of DLK-1, we were concerned that the observed effects could be confounded by the variable levels of overexpression associated with multicopy extrachromosomal

transgenes. We therefore generated single-copy transgene expressing DLK-1S or DLK-1L driven by the rgef-1 panneural promoter, using the Mos-SCI technique ( Frøkjaer-Jensen et al., 2008) ( Experimental Procedures). Single-copy expression of DLK-1S (juSi46) in wild-type animals significantly impaired PLM neuron axon regeneration, while single-copy expression of DLK-1L (juSi50) strongly enhanced regeneration in wild-type and in dlk-1(tm4024) mutants that lack both DLK-1L and DLK-1S ( Figure 6C). Importantly, expression of DLK-1L from juSi50 showed weak rescue of the failure of axon regeneration in dlk-1(ju476) mutants, which express intact DLK-1S ( Figures S1D and 6C). These results not only reaffirm that DLK-1S has potent and specific antagonistic effects on DLK-1L in axon regeneration, but also suggest that axonal injury can trigger DLK-1L activation by releasing the endogenous inhibition imposed by DLK-1S.