ULK1 (unc51-like autophagy activating kinase 1) is a serine/threonine kinase that plays a key role in regulating macroautophagy/autophagy induction in response to amino acid starvation. Despite the recent progress in understanding ULK1 functions, the molecular mechanism by which ULK1 regulates the induction of autophagy remains elusive. In this study, we determined that ULK1 phosphorylates Ser30 of BECN1 (Beclin 1) in association with ATG14 (autophagy-related 14) but not with UVRAG (UV radiation resistance associated). The Ser30 phosphorylation was induced by deprivation of amino acids or treatments with Torin 1 or rapamycin, the conditions that inhibit MTORC1 (mechanistic target of rapamycin complex 1), and requires ATG13 and RB1CC1 (RB1 inducible coiled-coil 1), proteins that interact with ULK1. Hypoxia or glutamine deprivation, which inhibit MTORC1, was also able to increase the phosphorylation in a manner dependent upon ULK1 and ULK2. Blocking the BECN1 phosphorylation by replacing Ser30 with alanine suppressed the amino acid starvation-induced activation of the ATG14-containing PIK3C3/VPS34 (phosphatidylinositol 3-kinase catalytic subunit type 3) kinase, and reduced autophagy flux and the formation of phagophores and autophagosomes. The Ser30-to-Ala mutation did not affect the ULK1-mediated phosphorylations of BECN1 Ser15 or ATG14 Ser29, indicating that the BECN1 Ser30 phosphorylation might regulate autophagy independently of those 2 sites. Taken together, these results demonstrate that BECN1 Ser30 is a ULK1 target site whose phosphorylation activates the ATG14-containing PIK3C3 complex and stimulates autophagosome formation in response to amino acid starvation, hypoxia, and MTORC1 inhibition.
Bibliographical noteFunding Information:
HHS j NIH j National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS); HHS j NIH j National Institute on Aging (NIA); HHS j NIH j National Institute of General Medical Sciences (NIGMS). This study was supported by the functional proteomics of aging NIH training grant [grant number T32AG029796 (to DG)], [grant number E0160500-02 (to CHJ)], [grant number AR055685 (to MK)], [grant number P30-DK050456] and [grant number GM097057 (to DHK)].
HHS | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS); HHS | NIH | National Institute on Aging (NIA); HHS | NIH | National Institute of General Medical Sciences (NIGMS). We thank M. Kundu for Ulk1 and Ulk2 MEFs; J.-L. Guan for Rb1cc1 KO MEFs; U87 cells for Z. Dong; L. Anderson in the Center for Mass Spectrometry and Proteomics for sample prep and analysis; R. Foncea at the Minnesota Obesity Center for lentivirus preparation; Kim lab and Neufeld lab members for discussion and helpful comments.
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