Previous studies have shown that the Regulator of Presynaptic Morphology (RPM-1) regulates synapse formation, axon guidance, and axon termination. RPM-1 functions as an ubiquitin ligase to negatively regulate a MAPK pathway that includes DLK-1, MKK-4 and the
p38 MAP kinase, PMK-3. The ubiquitin ligase activity of RPM-1 requires the F-box protein, FSN-1. RPM-1 is also a positive regulator of a Rab GTPase pathway that includes
glo-4 and
glo-1. While RPM-1 is the only known negative regulator of the DLK-1 pathway, studies in yeast and mammals have shown that Protein Phosphatase 2C (PP2C) a and b isoforms can negatively regulate MAPK pathways. C. elegans has a single homolog of mammalian PP2Ca and b that we call Protein Phosphatase Magnesium/manganese-dependent (PPM)-1 (previously called TAG-93). Here we test the role of
ppm-1 in axon termination and synapse formation using two loss-of-function (lf) alleles (
ok578 and
tm653).
ok578 and
tm653 are molecular null alleles as they both delete large amounts of the
ppm-1 gene including sequence encoding conserved residues that are essential for PP2C phosphatase activity.
ppm-1 (lf) mutants have defects in axon termination similar to mutants of molecules in the
rpm-1 pathway, including
fsn-1,
glo-4 and
glo-1. In
ppm-1;
fsn-1 double mutants axon termination defects are significantly enhanced demonstrating that
ppm-1 and
fsn-1 function in parallel pathways.
The presynaptic terminals of GABAergic motor neurons can be visualized using SNB-1::GFP, which localizes to evenly distributed puncta along the dorsal cord in wild-type animals. While the organization of SNB-1::GFP puncta is normal in
ppm-1 (lf) mutants,
ppm-1;
fsn-1 double mutants have significantly enhanced defects in organization and numbers of puncta. In contrast,
ppm-1,
rpm-1 double mutants are not enhanced. These results indicate that
ppm-1 functions in a parallel pathway to
fsn-1, and in the same pathway as
rpm-1.
We have found that
ppm-1 functions through its phosphatase activity downstream of
rpm-1, which suggests that PPM-1 may dephosphorylate and inactivate a kinase in the DLK-1 pathway. To test this hypothesis, we generated double mutants of
ppm-1 with
dlk-1,
mkk-4, or
pmk-3. Only
ppm-1;
pmk-3 double mutants were suppressed when compared to
ppm-1 (lf) mutants. Further, defects caused by transgenic overexpression of DLK-1 were rescued by coexpression of PPM-1. Overall, our results show that PPM-1 regulates axon termination and synapse formation by negatively regulating the DLK-1 pathway at the level of PMK-3.