Identifying Neurofibromin-Specific Regulatory Nodes for Therapeutic Targeting in NF1

The overall goal of the project is to determine how neurofibromin is regulated, and to explore the hypothesis that loss of neurofibromin activity leads to upregulation of specific receptors. We are building on our earlier discovery, that neurofibromin depends on the adapter protein SPRED1, to function, and we are utilizing the latest technical innovations including CRISPR technology to find genes that regulate neurofibromin SPRED function. To date we have demonstrated that oncogenic EGFR signaling disrupts Spred1NF1 binding. Mass spectrometry was performed on cells overexpressing EGFRL858R to identify potential phosphorylation sites on Spred1 and NF1 that could disrupt Spred1NF1 binding by steric hindrance. A serine phosphorylation site on Spred1 was identified in which a phosphomimetic and phosphodeficient mutant decreased or increased Spred1NF1 binding, respectively. Therefore, phosphorylation of Spred1 at this site by a serine kinase downstream of oncogenic EGFR may disrupt Spred1NF1 binding. Our findings provide one potential mechanism by which oncogenic EGFR signaling disrupts negative feedback to allow for constitutive Ras signaling. Furthermore, this work may elucidate a novel kinase therapeutic target for restoring NF1 mediated inhibition of Ras.