The Pam/Highwire/RPM-1 (PHR) proteins are key regulators of neuronal development that function in axon extension and guidance, termination of axon outgrowth, and synapse formation. Outside of development, the PHR proteins also regulate axon regeneration and Wallerian degeneration. The PHR proteins function in part by acting as ubiquitin ligases that degrade the Dual Leucine zipper-bearing Kinase (DLK). Here, we show that the Caenorhabditis elegans PHR protein, Regulator of Presynaptic Morphology 1 (RPM-1), also utilizes a phosphatase-based mechanism to regulate DLK-1. Using mass spectrometry, we identified Protein Phosphatase Magnesium/Manganese dependent 2 (PPM-2) as a novel RPM-1 binding protein. Genetic, transgenic, and biochemical studies indicated that PPM-2 functions coordinately with the ubiquitin ligase activity of RPM-1 and the F-box protein FSN-1 to negatively regulate DLK-1. PPM-2 acts on S874 of DLK-1, a residue implicated in regulation of DLK-1 binding to a short, inhibitory isoform of DLK-1 (DLK-1S). Our study demonstrates that PHR proteins function through both phosphatase and ubiquitin ligase mechanisms to inhibit DLK. Thus, PHR proteins are potentially more accurate and sensitive regulators of DLK than originally thought. Our results also highlight an important and expanding role for the PP2C phosphatase family in neuronal development.

Mutations in Nesprin-1 and 2 (also called Syne-1 and 2) are associated with numerous diseases including autism, cerebellar ataxia, cancer, and Emery-Dreifuss muscular dystrophy. Nesprin-1 and 2 have conserved orthologs in flies and worms called MSP-300 and abnormal nuclear Anchorage 1 (ANC-1), respectively. The Nesprin protein family mediates nuclear and organelle anchorage and positioning. In the nervous system, the only known function of Nesprin-1 and 2 is in regulation of neurogenesis and neural migration. It remains unclear if Nesprin-1 and 2 regulate other functions in neurons. Using a proteomic approach in C. elegans, we have found that ANC-1 binds to the Regulator of Presynaptic Morphology 1 (RPM-1). RPM-1 is part of a conserved family of signaling molecules called Pam/Highwire/RPM-1 (PHR) proteins that are important regulators of neuronal development. We have found that ANC-1, like RPM-1, regulates axon termination and synapse formation. Our genetic analysis indicates that ANC-1 functions via the β-catenin BAR-1, and the ANC-1/BAR-1 pathway functions cell autonomously, downstream of RPM-1 to regulate neuronal development. Further, ANC-1 binding to the nucleus is required for its function in axon termination and synapse formation. We identify variable roles for four different Wnts (LIN-44, EGL-20, CWN-1 and CWN-2) that function through BAR-1 to regulate axon termination. Our study highlights an emerging, broad role for ANC-1 in neuronal development, and unveils a new and unexpected mechanism by which RPM-1 functions.

Background: How RPM-1 interacts with FSN-1 remains unknown. Results: Structure-function and transgenic analysis define the biochemical relationship between RPM-1 and FSN-1. Conclusion: RPM-1 uses a conserved mechanism to bind FSN-1 that is independent of RPM-1 ubiquitin ligase activity. Significance: Our biochemical and genetic analysis has led to identification of RIP, an in vivo inhibitor of the RPM-1⅐FSN-1 ubiquitin ligase complex.

We show that loss-of-function mutations in kinases of the MLK-1 pathway (mlk-1, mek-1, and kgb-1/jnk) function cellautonomously in neurons to suppress defects in synapse formation and axon termination caused by rpm-1 loss of function. Our genetic analysis also suggests that the phosphatase PPM-1, like RPM-1, is a potential inhibitor of kinases in the MLK-1 pathway. IN Caenorhabditis elegans, the ubiquitin ligase Regulator of Presynaptic Morphology 1 (RPM-1) regulates neuronal development by inhibiting the DLK-1 pathway (composed of DLK-1, MKK-4, and PMK-3) (Nakata et al. 2005). RPM-1 and the DLK-1 pathway regulate axon regeneration postdevelopmentally (Hammarlund et al. 2009;Yan et al. 2009). The MLK-1 pathway, which includes the kinases MLK-1, MEK-1, and KGB-1/JNK, also regulates axon regeneration (Nix et al. 2011). It remains unclear if RPM-1 functions through the MLK-1 pathway to regulate development.Protein Phosphatase Mg/Mn2+-dependent 1 (PPM-1) and PPM-2 negatively regulate the DLK-1 pathway (Tulgren et al. 2011;Baker et al. 2014). PPM-2 is regulated by RPM-1 and dephosphorylates full-length DLK-1 (DLK-1L). PPM-1 is likely to function lower in the DLK-1 pathway. It remains unclear whether PPM-1 and PPM-2 regulate other signaling pathways in the neurons of C. elegans.Here, we show that mutations in mlk-1, mek-1, and kgb-1/ jnk suppress defects in synapse formation and axon termination caused by rpm-1 loss of function (lf). These results suggest that RPM-1 might negatively regulate the MLK-1 pathway, which is consistent with our observation that transgenic overexpression of MLK-1 or KGB-1 caused axon termination defects. Furthermore, our results are consistent with PPM-1 negatively regulating the MLK-1 pathway, in addition to inhibiting the DLK-1 pathway. In contrast, our findings suggest that PPM-2 acts only on DLK-1L. ResultsLoss-of-function mutations in kinases of the MLK-1 pathway suppress defects in synapse formation caused by rpm-1 (lf)The fly ortholog of RPM-1, called Highwire, functions through JNK to regulate synapse formation (Collins et al. 2006). It is unclear if RPM-1 functions through JNK to regulate synapse formation in worms, but studies on axon regeneration suggested that this might be a possibility (Nix et al. 2011). Hence, we assessed the genetic relationship between kinases of the MLK-1 pathway, including kgb-1/jnk and rpm-1 in the context of synapse formation.Consistent with previous studies, the GABAergic motor neurons of rpm-12/2 mutants had synapse formation defects that were strongly, but incompletely, suppressed in rpm-12/2; dlk-12/2 double mutants ( Figure 1, A and B) (Nakata et al. 2005). In double mutants of rpm-1 with mlk-1, mek-1, or kgb-1, significant but modest suppression occurred (Figure 1, A 1B). rpm-12/2 mlk-12/2; dlk-1 triple mutants showed a small, but significant, increase in suppression, consistent with mlk-1 and dlk-1 functioning in partially redundant pathways ( Figure 1B). These results are consistent with RPM-1 regulating synapse formation by inhibiting ...