Abstract
The hominoid oncogene TBC1D3 enhances epidermal growth factor receptor (EGFR) signaling and induces cell transformation. However, little is known regarding its spatio-temporal regulation and mechanism of tumorigenesis. In the current study, we identified the microtubule subunit β-tubulin as a potential interaction partner for TBC1D3 using affinity purification combined with mass spectrometry analysis. The interaction between TBC1D3 and β-tubulin was confirmed by co-immunoprecipitation. Using the same method, we also revealed that TBC1D3 co-precipitated with endogenous α-tubulin, another subunit of the microtubule. In agreement with these results, microtubule cosedimentation assays showed that TBC1D3 associated with the microtubule network. The β-tubulin-interacting site of TBC1D3 was mapped to amino acids 286∼353 near the C-terminus of the TBC domain. Deletion mutation within these amino acids was shown to abolish the interaction of TBC1D3 with β-tubulin. Interestingly, the deletion mutation caused a complete loss of TBC1D3 from the cytoplasmic filamentous and punctate structures, and TBC1D3 instead appeared in the nucleus. Consistent with this, wild-type TBC1D3 exhibited the same nucleocytoplasmic distribution in cells treated with the microtubule depolymerizing agent nocodazole, suggesting that the microtubule network associates with and retains TBC1D3 in the cytoplasm. We further found that deficiency in β-tubulin-interacting resulted in TBC1D3's inability to inhibit c-Cbl recruitment and EGFR ubiquitination, ultimately leading to dysregulation of EGFR degradation and signaling. Taken together, these studies indicate a novel model by which the microtubule network regulates EGFR stability and signaling through tubulin dimer/oligomer interaction with the nucleocytoplasmic protein TBC1D3.
Highlights
The epidermal growth factor receptor (EGFR) is the first identified member of the ErbB receptor tyrosine kinase family
TBC1D3 interacts with b-tubulin To gain insights into TBC1D3 function and regulation, we sought to identify its interaction partners using a GST pull-down assay combined with mass spectrometry analysis
Our studies identify tubulin/microtubule network as a novel regulator of TBC1D3
Summary
The epidermal growth factor receptor (EGFR) is the first identified member of the ErbB receptor tyrosine kinase family. As one of the most common post-translational modifications, ubiquitination of EGFR plays a critical role in endocytic trafficking and lysosomal degradation of the activated receptor. The alternative mechanism involves the tyrosine residues Tyr 1068 and Tyr 1086 of the activated receptor, which indirectly recruits Cbl through its interaction with the SH3 domain of Grb2 [8]. With Cbl serving as an adaptor to bridge Ubc4/5 E2 ubiquitin-conjugating enzyme interaction with EGFR, ubiquitin is transferred directly from the E2 to distinct lysine residues within the kinase domain of EGFR, including six major ubiquitin conjugation sites (Lys692, Lys713, Lys, 730, Lys843, Lys905, and Lys946) [9,10]. Sufficient but not essential for EGFR internalization [11,12,13], EGFR ubiquitination is required for its sorting onto intraluminal vesicles of multivesicular endosomes/bodies and subsequent lysosomes for efficient degradation [12,14]
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