RPM-1 Uses Both Ubiquitin Ligase and Phosphatase-Based Mechanisms to Regulate DLK-1 during Neuronal Development

Abstract

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.

Author Summary

The molecular mechanisms that govern formation of functional synaptic connections are central to brain development and function. We have used the nematode C. elegans to explore the mechanism of how the intracellular signaling protein RPM-1 regulates neuronal development. Using a combination of proteomic, genetic, transgenic, and biochemical approaches we have shown that RPM-1 functions through a PP2C phosphatase, PPM-2, to regulate the activity of a MAP3 kinase, DLK-1. Our results indicate that a combination of PPM-2 phosphatase activity and RPM-1 ubiquitin ligase activity inhibit DLK-1.