The evolutionarily conserved domain of Beclin 1 is required for Vps34 binding, autophagy and tumor suppressor function.
Atg6/Beclin 1 is an evolutionarily conserved protein family that has been shown to function in vacuolar protein sorting (VPS) in yeast; in autophagy in yeast, Drosophila, Dictyostelium, C.elegans, and mammals; and in tumor suppression in mice. Atg6/Beclin 1 is thought to function as a VPS and autophagy protein as part of a complex with Class III phosphatidylinositol 3'-kinase (PI3K)/Vps34. However, nothing is known about which domains of Atg6/Beclin 1 are required for its functional activity and binding to Vps34. We hypothesized that the most highly conserved region of human Beclin 1 spanning from amino acids 244-337 is essential for Vps34 binding, autophagy, and tumor suppressor function. To investigate this hypothesis, we evaluated the effects of wild-type and mutant beclin 1 gene transfer in autophagy-deficient MCF7 human breast carcinoma cells. We found that, unlike wild-type Beclin 1, a Beclin 1 mutant lacking aa 244-337 (Beclin 1DeltaECD), is unable to enhance starvation-induced autophagy in low Beclin 1-expressing MCF7 human breast carcinoma cells. In contrast to wild-type Beclin 1, mutant Beclin 1DeltaECD is unable to immunoprecipitate Vps34, has no Beclin 1-associated Vps34 kinase activity, and lacks tumor suppressor function in an MCF7 scid mouse xenograft tumor model. The maturation of cathepsin D, which requires intact Vps34-dependent VPS function, is comparable in autophagy-deficient low-Beclin 1 expressing MCF7 cells, autophagy-deficient MCF7 cells transfected with Beclin 1DeltaECD, and autophagy-competent MCF7 cells transfected with wild-type Beclin 1. These findings identify an evolutionarily conserved domain of Beclin 1 that is essential for Vps34 interaction, autophagy function, and tumor suppressor function. Furthermore, they suggest a connection between Beclin 1-associated Class III PI3K/Vps34-dependent autophagy, but not VPS, function and the mechanism of Beclin 1 tumor suppressor action in human breast cancer cells.
Furuya
Norihiko
N
Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York, USA.
Yu
Jie
J
Byfield
Maya
M
Pattingre
Sophie
S
Levine
Beth
B
D000595:Amino Acid Sequence; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000502:physiology*; D001343:Autophagy / Q000502:physiology*; D000071186:Beclin-1; D002402:Cathepsin D / Q000378:metabolism; D045744:Cell Line, Tumor; D017124:Conserved Sequence; D029721:Drosophila Proteins / Q000235:genetics / Q000502:physiology*; D019143:Evolution, Molecular; D016147:Genes, Tumor Suppressor; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D009368:Neoplasm Transplantation; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014183:Transplantation, Heterologous; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000502:physiology*
D051017:Apoptosis Regulatory Proteins; C580769:Atg6 protein, Drosophila; C491997:BECN1 protein, human; D000071186:Beclin-1; D029721:Drosophila Proteins; D008565:Membrane Proteins; D033921:Vesicular Transport Proteins; D019869:Phosphatidylinositol 3-Kinases; D002402:Cathepsin D