Zhong Y, Wang QJ, Li XT, Yan Y, Backer JM, Chait BT et al. Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex. Nat Cell Biol 11: 468-476

Nature Cell Biology (Impact Factor: 19.68). 05/2009; 11(4):468-76. DOI: 10.1038/ncb1854
Source: PubMed


Beclin 1, a mammalian autophagy protein that has been implicated in development, tumour suppression, neurodegeneration and cell death, exists in a complex with Vps34, the class III phosphatidylinositol-3-kinase (PI(3)K) that mediates multiple vesicle-trafficking processes including endocytosis and autophagy. However, the precise role of the Beclin 1-Vps34 complex in autophagy regulation remains to be elucidated. Combining mouse genetics and biochemistry, we have identified a large in vivo Beclin 1 complex containing the known proteins Vps34, p150/Vps15 and UVRAG, as well as two newly identified proteins, Atg14L (yeast Atg14-like) and Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein). Characterization of the new proteins revealed that Atg14L enhances Vps34 lipid kinase activity and upregulates autophagy, whereas Rubicon reduces Vps34 activity and downregulates autophagy. We show that Beclin 1 and Atg14L synergistically promote the formation of double-membraned organelles that are associated with Atg5 and Atg12, whereas forced expression of Rubicon results in aberrant late endosomal/lysosomal structures and impaired autophagosome maturation. We hypothesize that by forming distinct protein complexes, Beclin 1 and its binding proteins orchestrate the precise function of the class III PI(3)K in regulating autophagy at multiple steps.

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    • "vector for mammalian expression. Sec3/5/6/8/10/15/70-Flag (Thapa et al., 2012), Myc-Exo84 (Moskalenko et al., 2003), HA-Sec5 (Moskalenko et al., 2002) and Flag-Rubicon (Zhong et al., 2009) were described previously. For expression of EGFR-Flag by lentivirus, EGFR ORF from pCDNA-EGFR-WT and -KD (SJ Parsons, University of Virginia) was cloned into pWPT-GFP vector with the GFP ORF replaced by EGFR ORF. "
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    ABSTRACT: The epidermal growth factor receptor (EGFR) is upregulated in numerous human cancers. Inhibition of EGFR signaling induces autophagy in tumor cells. Here, we report an unanticipated role for the inactive EGFR in autophagy initiation. Inactive EGFR interacts with the oncoprotein LAPTM4B that is required for the endosomal accumulation of EGFR upon serum starvation. Inactive EGFR and LAPTM4B stabilize each other at endosomes and recruit the exocyst subcomplex containing Sec5. We show that inactive EGFR, LAPTM4B, and the Sec5 subcomplex are required for basal and starvation-induced autophagy. LAPTM4B and Sec5 promote EGFR association with the autophagy inhibitor Rubicon, which in turn disassociates Beclin 1 from Rubicon to initiate autophagy. Thus, the oncoprotein LAPTM4B facilitates the role of inactive EGFR in autophagy initiation. This pathway is positioned to control tumor metabolism and promote tumor cell survival upon serum deprivation or metabolic stress. Copyright © 2015 Elsevier Inc. All rights reserved.
    Cell 01/2015; 160(1-2):145-160. DOI:10.1016/j.cell.2014.12.006 · 32.24 Impact Factor
    • "UVRAG competes with ATG14L for binding to the beclin 1-hVps34 complex and promotes autophagosome fusion with lysosomes[38]. Rubicon forms a complex with UVRAG-beclin 1-hVps34 and inhibits autophagy[37]. We co-expressed FLAG-Cu/Zn superoxide dismutase 1 WT or G93A with EGFP-vector or EGFP-beclin 1 in NSC34 cells, then 48 hours later used an anti-GFP antibody to co-immunoprecipitate the EGFP-tagged protein with its interacting proteins. "
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    ABSTRACT: Previous studies have confirmed that the beclin 1 complex plays a key role in the initial stage of autophagy and deregulated autophagy might involve in amyotrophic lateral sclerosis. However, the mechanism underlying altered autophagy associated with the beclin 1 complex remains unclear. In this study, we transfected the Cu/Zn superoxide dismutase 1 G93A mutant protein into the motor neuron-like cell line NSC34 cultured in vitro. Western blotting and co-immunoprecipitation showed that the Cu/Zn superoxide dismutase 1 G93A mutant enhanced the turnover of autophagic marker microtubule-associated protein light chain 3II (LC3II) and stimulated the conversion of EGFP-LC3I to EGFP-LC3II, but had little influence on the binding capacity of the autophagy modulators ATG14L, rubicon, UVRAG, and hVps34 to beclin 1 during autophagosome formation. These results suggest that the amyotrophic lateral sclerosis-linked Cu/Zn superoxide dismutase 1 G93A mutant can upregulate autophagic activity in NSC34 cells, but that this does not markedly affect beclin 1 complex components.
    Neural Regeneration Research 09/2014; 9(1):16-24. DOI:10.4103/1673-5374.125325 · 0.22 Impact Factor
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    • "The PI3K complex type 3 is formed by these complexes, and the resulting complex is involved in the nucleation step of autophagy. Therefore, the complex formation controls the activation level of autophagy through inhibition to combine the autophagosome and the lysosome in the complex by binding Rubicon [19]. However, the nucleation step occurs in the membranes of the endoplasmic reticulum or the mitochondria by the activation of the PI3K complex type 3. "
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    ABSTRACT: The placenta is a temporary fetomaternal organ capable of supporting fetal growth and development during pregnancy. In particular, abnormal development and dysfunction of the placenta due to cha nges in the proliferation, differentiation, cell death, and invasion of trophoblasts induce several gynecological diseases as well as abnormal fetal development. Autophagy is a catalytic process that maintains cellular structures by recycling building blocks derived from damaged microorganelles or proteins resulting from digestion in lysosomes. Additionally, autophagy is necessary to maintain homeostasis during cellular growth, development, and differentiation, and to protect cells from nutritional deficiencies or factors related to metabolism inhibition. Induced autophagy by various environmental factors has a dual role: it facilitates cellular survival in normal conditions, but the cascade of cellular death is accelerated by over-activated autophagy. Therefore, cellular death by autophagy has been known as programmed cell death type II. Autophagy causes or inhibits cellular death via the other mechanism, apoptosis, which is programmed cell death type I. Recently, it has been reported that autophagy increases in placenta-related obstetrical diseases such as preeclampsia and intrauterine growth retardation, although the mechanisms are still unclear. In particular, abnormal autophagic mechanisms prevent trophoblast invasion and inhibit trophoblast functions. Therefore, the objectives of this review are to examine the characteristics and functions of autophagy and to investigate the role of autophagy in the placenta and the trophoblast as a regulator of cell death.
    Clinical and Experimental Reproductive Medicine 09/2014; 41(3):97-107. DOI:10.5653/cerm.2014.41.3.97
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