Anti-BECN1 monoclonal antibody, clone 5B21

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is central to autophagy initiation. We previously reported the V-shaped architecture of the four-subunit version of PI3KC3-C1 consisting of VPS (vacuolar protein sorting) 34, VPS15, BECN1 (Beclin 1), and ATG (autophagy-related) 14. Here we show that a putative fifth subunit, nuclear receptor binding factor 2 (NRBF2), is a tightly bound component of the complex that profoundly affects its activity and architecture. NRBF2 enhances the lipid kinase activity of the catalytic subunit, VPS34, by roughly 10-fold. We used hydrogen-deuterium exchange coupled to mass spectrometry and negative-stain electron microscopy to map NRBF2 to the base of the V-shaped complex. NRBF2 interacts primarily with the N termini of ATG14 and BECN1. We show that NRBF2 is a homodimer and drives the dimerization of the larger PI3KC3-C1 complex, with implications for the higher-order organization of the preautophagosomal structure.

It has been shown that sphingosine-1-phosphate (S1P) is elevated in patients with pulmonary arterial hypertension (PAH) and promotes the proliferation of pulmonary artery smooth muscle cells (PASMCs). Meanwhile, S1P has been found to induce the activation of autophagy in several types of human diseases including cancers. However, it is still unclear whether activation of autophagy mediates S1P-induced PASMCs proliferation, and detailed mechanisms responsible for these processes are indefinite. The aims of this study are to address these issues. S1P doseand time-dependently reduced the expression of E-cadherin/CDH1 and stimulated PASMCs proliferation; this was accompanied with the elevation of TNF receptor-associated factor 2 (TRAF2), upregulation and ubiquitination of BECN1 and the activation of autophagy. Prior silencing TRAF2 or BECN1 using siRNA or pre-incubation of cells with autophagy inhibitor chloroquine phosphate (CQ) suppressed S1P-induced autophagy activation and subsequent CDH1 degradation and further PASMCs proliferation. Taken together, our study indicates that S1P promotes the activation of autophagy by accelerating TRAF2-mediated BECN1 up-regulation and ubiquitination, which in turn results in CDH1 reduction and contributes to PASMCs proliferation.