Dysregulated Intracellular Trafficking in Immortalized Human Fibroblasts from Noonan and CBL Syndrome Patients.

摘要

Noonan and related syndromes, the "RASopathies", are due to autosomal dominant mutations in Ras/MAPK pathway genes including ones encoding the protein tyrosine phosphatase SHP-2 (PTPN11), a Ras-GEF, SOS1, and CBL, a E3 ubiquitin ligase involved in receptor tyrosine kinase (RTK) degradation. The aim of this study was to investigate whether RASopathy mutations result in dysregulated RTK trafficking, contributing to excessive Ras/MAPK signaling. To study this, we developed immortalized skin fibroblast cell lines from individuals with PTPN11, SOS1, and CBL mutations. Wild-type and mutant cells were stimulated with fluorescently labeled EGF, and EGF internalization was tracked. Compared to wild-type cells, EGF internalization was significantly delayed in the RASopathies. Next, cell membrane proteins were biotinylated and loss of EGFR from the cell membrane after EGF stimulation was assessed. We observed that cell-membrane EGFR content remained higher in the RASopathies compared to wild type. Strikingly, SOS1 mutations resulted in greater delays in internalization than CBL and PTPN11 mutations. Taken together, our results show that RASopathy-associated mutations delay EGF-EGFR internalization, potentially prolonging Ras/MAPK signaling. Further analysis is required to track RTK-ligand complexes intracellularly in the endosomal compartments, and to determine which endocytotic mechanisms (clathrin-dependent vs. -independent) are relevant.