Connexin 31.1 degradation requires the Clathrin-mediated autophagy in NSCLC cell H1299.
Connexins have relative short half-lives. Connexin 31.1 (Cx31.1) was newly reported to be down-regulated in non-small cell lung cancer cell lines, and displayed tumour-suppressive properties. However, no reports describing how a cell regulates Cx31.1 level were found. In this study, Cx31.1 was suggested to be degraded through both ubiquitin-proteasome system (UPS) and autophagy. Blockage of UPS with MG-132 increased Cx31.1 level, but could not inhibit the degradation of Cx31.1 completely. In H1299 cells stably expressing Cx31.1, Cx31.1 reduced when autophagy was induced through starvation or Brefeldin A treatment. Knockdown of autophagy-related protein ATG5 could increase the cellular level of Cx31.1 both under normal growth condition and starvation-induced autophagy. Colocalization of Cx31.1 and autophagy marker light chain 3 (LC3) was revealed by immunofluorescence analysis. Coimmunoprecipitation and immunofluorescence showed that Cx31.1 might interact with clathrin heavy chain which was newly reported to regulate autophagic lysosome reformation (ALR) and controls lysosome homoeostasis. When clathrin expression was knockdown by siRNA treatment, the level of Cx31.1 increased prominently both under normal growth condition and starvation-induced autophagy. Under starvation-induced autophagy, LC3-II levels were slightly accumulated with siCla. treatment compared to that of siNC, which could be ascribed to that clathrin knockdown impaired the late stage of autophagy, ALR. Taken together, we found autophagy contributed to Cx31.1 degradation, and clathrin might be involved in the autophagy of Cx31.1.
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, China.
Xingli
X
Zhu
Zhenchao
Z
Ruan
Xiufang
X
Yang
Kaili
K
Chu
Hai
H
Wu
Yao
Y
Li
Yan
Y
Huang
D001343:Autophagy; D000071187:Autophagy-Related Protein 5; D015415:Biomarkers / Q000378:metabolism; D020126:Brefeldin A / Q000494:pharmacology; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002966:Clathrin / Q000378:metabolism*; D017630:Connexins / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism
C502778:ATG5 protein, human; D000071187:Autophagy-Related Protein 5; D015415:Biomarkers; D002966:Clathrin; D017630:Connexins; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; C076274:connexin 31.1; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; D046988:Proteasome Endopeptidase Complex
Connexin 31.1; autophagy; clathrin; starvation; ubiquitin-proteasome system