Summary The balance of protein phosphorylation is essential for normal cellular development and physiology. Disruption of this balance may lead to diseases such as cancer, diabetes, inflammation, and Alzheimer’s. Kinases and phosphatases are the enzymes that maintain this balance. There are atypical members of these superfamilies that lack catalytic activity (pseudoenzymes), but maintain their structure, at least some of which serve as signaling molecules. In particular, the pseudophosphatase MK-STYX elicits neurite formation, which is the initiation of neuron differentiation, in multiple neuronal cell models. The identification and characterization of molecules with which MK-STYX interacts and regulates in these neuronal models are essential to understand how MK-STYX serves as a signaling regulator in cellular differentiation. Our prior studies show that MK-STYX elicits neurite extensions and decreases activation of the protein RhoA in a neuronal model (rat PC12 cells). However, how MK-STYX regulates these dynamics remains unknown. To fill this gap in our understanding, we will investigate the role of MK-STYX as a signal regulator in multiple neuronal models (human, rat and mouse cultured cells and rat primary hippocampal neurons). We will investigate MK-STYX’s and MK-STYX-mutants’ effects on neurite formation in cultured cells and development of primary neurons. Intriguingly, it was recently reported that MK-STYX interacts with several cytoskeletal proteins. Given that MK-STYX enhances primary and secondary neurites in PC12 cells, we hypothesize that MK-STYX promotes primary neuron development through interaction with cytoskeletal proteins. We will use the combined approach of overexpression studies, CRSPR Cas or RNA interference (RNAi), immunofluorescence and mass spectrometry. Investigating MK-STYX’ various domains (mutants) in neurite formation in various neuronal models, interactomes (interacting partners) in multiple neuronal models, and its role in primary neurons is an important unexplored dimension. Results of the proposed study will provide a mechanism of MK-STYX’s mode of action, which will allow the development of a mechanistic model. In addition, it will increase our understanding of MK-STYX in the regulation of neuronal development. Our goal is to provide insight into this pseudophosphatase as a signaling molecule in neuronal cell signaling pathways.