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

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.Pam/Highwire/RPM-1（PHR）蛋白是神经元发育的关键调控因子，参与轴突延伸、轴突导向、轴突生长终止及突触形成等过程。在发育阶段之外，PHR蛋白还可调控轴突再生与沃勒变性（Wallerian degeneration）。PHR蛋白的部分功能通过充当泛素连接酶，降解双亮氨酸拉链激酶（Dual Leucine zipper-bearing Kinase，DLK）来实现。本研究发现，秀丽隐杆线虫（Caenorhabditis elegans）的PHR蛋白——突触形态调节因子1（Regulator of Presynaptic Morphology 1，RPM-1），还可通过磷酸酶依赖机制对DLK-1进行调控。我们借助质谱分析法（mass spectrometry）鉴定出镁/锰依赖型蛋白磷酸酶2（Protein Phosphatase Magnesium/Manganese-dependent 2，PPM-2）为新型RPM-1结合蛋白。遗传学、转基因及生化实验结果显示，PPM-2与RPM-1的泛素连接酶活性及F盒蛋白FSN-1协同作用，对DLK-1产生负向调控。PPM-2作用于DLK-1的S874位点，该位点的调控与DLK-1结合其短片段抑制性剪接变体DLK-1S密切相关。本研究证实，PHR蛋白可同时通过磷酸酶与泛素连接酶两种机制抑制DLK。由此表明，PHR蛋白对DLK的调控精准度与灵敏度或远超此前预期。本研究结果同时凸显了PP2C磷酸酶家族（PP2C phosphatase family）在神经元发育中重要且持续拓展的功能。