Cytoplasmic retention of a nucleocytoplasmic protein TBC1D3 by microtubule network is required for enhanced EGFR signaling.
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.
Department of Pathology and Pathophysiology, Medical School, Southeast University, Nanjing, Jiangsu, People's Republic of China; Key Laboratory of Developmental Genes and Human Diseases, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing, Jiangsu, People's Republic of China.
Ze
Z
He
Department of Pathology and Pathophysiology, Medical School, Southeast University, Nanjing, Jiangsu, People's Republic of China.
Tian
T
Tian
Department of Pathology and Pathophysiology, Medical School, Southeast University, Nanjing, Jiangsu, People's Republic of China.
Dan
D
Guo
Department of Pathology and Pathophysiology, Medical School, Southeast University, Nanjing, Jiangsu, People's Republic of China.
Huijuan
H
Wu
Department of Pathology and Pathophysiology, Medical School, Southeast University, Nanjing, Jiangsu, People's Republic of China.
Yang
Y
Chen
Department of Pathology and Pathophysiology, Medical School, Southeast University, Nanjing, Jiangsu, People's Republic of China.
Yongchen
Y
Zhang
Department of Pathology and Pathophysiology, Medical School, Southeast University, Nanjing, Jiangsu, People's Republic of China.
Qing
Q
Wan
Department of Pathology and Pathophysiology, Medical School, Southeast University, Nanjing, Jiangsu, People's Republic of China.
Huzi
H
Zhao
Department of Pathology and Pathophysiology, Medical School, Southeast University, Nanjing, Jiangsu, People's Republic of China.
Congyang
C
Wang
Department of Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America.
Hongjing
H
Shen
Department of Pathology and Pathophysiology, Medical School, Southeast University, Nanjing, Jiangsu, People's Republic of China.
Lei
L
Zhao
Department of Pathology and Pathophysiology, Medical School, Southeast University, Nanjing, Jiangsu, People's Republic of China.
Xiaodong
X
Bu
Department of Pathology and Pathophysiology, Medical School, Southeast University, Nanjing, Jiangsu, People's Republic of China.
Meiling
M
Wan
Department of Pathology and Pathophysiology, Medical School, Southeast University, Nanjing, Jiangsu, People's Republic of China.
Chuanlu
C
Shen
D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000502:physiology; D003593:Cytoplasm / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006801:Humans; D008870:Microtubules / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014404:Tubulin / Q000378:metabolism
D020690:GTPase-Activating Proteins; D011518:Proto-Oncogene Proteins; C467563:TBC1D3 protein, human; D014404:Tubulin; C512478:EGFR protein, human; D066246:ErbB Receptors