Dataset Information

ULK1/2 DKO MEF phosphoproteomics

ABSTRACT: Autophagy is a mechanism by which intracellular cargo are catabolised in the lysosome. It involves the formation of double membraned organelles termed autophagosomes, a complex process orchestrated by the serine/threonine kinase ULK complex and class III PI3 kinase VPS34. Unbiased screens for ULK substrates revealed that the phosphoproteome is significantly altered upon loss of Ulk1/2 and yielded a high confidence list of substrates, including VPS34 complex member VPS15 and AMPK complex member PRKAG2. We identified 6 new ULK substrate residues in the VPS15. Mutation of these VPS15 phosphoacceptors reduces autophagosome formation and autophagic flux in cells, and VPS34 activity in vitro. ULK phosphorylation of the major phosphosite in VPS15, serine 861, decreases both autophagy initiation and autophagic flux. Analysis of VPS15 knockout cells and the ULK substrate VPS34 serine 249 revealed two novel ULK-dependent phenotypes downstream of VPS15 removal: starvation-independent accumulation of ULK substrates and kinase activity-regulated recruitment of autophagy proteins to ubiquitin-positive structures, both of which occur upon VPS15 ablation and can be partially recapitulated by chronic VPS34 inhibition.

INSTRUMENT(S): Orbitrap Fusion Lumos, LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Helen Flynn

LAB HEAD: Bram Snijders

PROVIDER: PXD022228 | Pride | 2021-06-15

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	SPW572857406484txtSPW6484-40-502898.zip	Other
	SPW5728A1RW30_HJ_A1SPW5728A1RW30-40-469636.raw	Raw
	SPW5728A1RW31_HJ_A1SPW5728A1RW31-40-469634.raw	Raw
	SPW5728A1RW32_HJ_A1SPW5728A1RW32-40-469635.raw	Raw
	SPW5728A1RW33_HJ_B1SPW5728A1RW33-40-469653.raw	Raw

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Publications

Phosphoproteomic identification of ULK substrates reveals VPS15-dependent ULK/VPS34 interplay in the regulation of autophagy.

Mercer Thomas John TJ Ohashi Yohei Y Boeing Stefan S Jefferies Harold B J HBJ De Tito Stefano S Flynn Helen H Tremel Shirley S Zhang Wenxin W Wirth Martina M Frith David D Snijders Ambrosius P AP Williams Roger Lee RL Tooze Sharon A SA

The EMBO journal 20210614 14

Autophagy is a process through which intracellular cargoes are catabolised inside lysosomes. It involves the formation of autophagosomes initiated by the serine/threonine kinase ULK and class III PI3 kinase VPS34 complexes. Here, unbiased phosphoproteomics screens in mouse embryonic fibroblasts deleted for Ulk1/2 reveal that ULK loss significantly alters the phosphoproteome, with novel high confidence substrates identified including VPS34 complex member VPS15 and AMPK complex subunit PRKAG2. We ...[more]

PMID: 34121209

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