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Phosphosite mapping on PI3Kc1 by ULK1 and TBK1 kinases.

ABSTRACT: Autophagy is a highly conserved catabolic process which degrades and recycles intracellular components. To accomplish this task the surplus material (cargo) is sequestered in a double membranous vesicle termed autophagosome. To initiate the formation of autophagosome autophagy machinery is recruited to cargo in a controlled manner. One of the first factors recruited to damaged mitochondria during mitophagy (selective removal of damaged mitochondria by autophagy) are two protein kinases, ULK1/2 complex and TBK1. They both promote a subsequent recruitment of the class III phosphatidylinositol 3-kinase complex I (PI3Kc1 complex) which function is to generate the lipid phosphatidylinositol 3-phosphate (PI(3)P) on phagophore membranes. Both ULK1 and TBK1 were reported to phosphorylate several autophagy factors. Here, we probed phosphorylation of PI3Kc1 by ULK1 and TBK1 to gain a full picture of the phosphosites map of PI3Kc phosphorylated by both protein kinases. Recombinant PI3Kc1 was incubated with recombinant ULK1 complex or TBK1 in the presence of ATP and MgCl2. As a negative control, the same protein mixtures were incubated in a kinase buffer lacking MgCl2 and ATP. Samples were separated by SDS-PAGE and stained with Coomassie. The bands for each subunit of the PI3KC3-C1 complex (VPS15, VPS34, ATG14, and Beclin1) were extracted from the gel and submitted for mass spectrometry analysis.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: WeiQiang Chen

LAB HEAD: Sascha Martens

PROVIDER: PXD039663 | Pride | 2023-05-08

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	2021.04_uniprot-taxonomy_7108_Spodoptera_frugiperda.fasta	Fasta
	20211203_QExHFX2_RSLC10_Bernklau_Martens_MPL_UniWien_TBK1_ATG14_Becline_0_s3_50p.raw	Raw
	20211203_QExHFX2_RSLC10_Bernklau_Martens_MPL_UniWien_TBK1_ATG14_Becline_30_s6_50p.raw	Raw
	20211203_QExHFX2_RSLC10_Bernklau_Martens_MPL_UniWien_TBK1_VPS15_0_s1_50p.raw	Raw
	20211203_QExHFX2_RSLC10_Bernklau_Martens_MPL_UniWien_TBK1_VPS15_30_s4_50p.raw	Raw

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Project description:Removal of damaged organelles via the process of selective autophagy constitutes a major form of cellular quality control. Damaged organelles are recognized by a dedicated surveillance machinery, leading to the assembly of an autophagosome around the damaged organelle, prior to fusion with the degradative lysosomal compartment. Lysosomes themselves are also prone to damage and are degraded through the process of lysophagy. While early steps involve recognition of ruptured lysosomal membranes by glycan-binding Galectins and ubiquitylation of transmembrane lysosomal proteins, many steps in the process, and their inter-relationships, remain poorly understood, including the role and identity of cargo receptors required for completion of lysophagy. Here, we employ quantitative organelle capture and proximity biotinylation proteomics of autophagy adaptors, cargo receptors, and Galectins in response to acute lysosomal damage, thereby revealing the landscape of lysosomal proteome remodeling during lysophagy. Among proteins dynamically recruited to damaged lysosomes were ubiquitin-binding autophagic cargo receptors. Using newly developed lysophagic flux reporters including Lyso-Keima, we demonstrate that TAX1BP1, together with its associated kinase TBK1, are both necessary and sufficient to promote lysophagic flux in both Hela cells and induced neurons (iNeurons). While the related receptor OPTN can drive damage-dependent lysophagy when overexpressed, cells lacking either OPTN or CALCOCO2 still maintain significant lysophagic flux in HeLa cells. Mechanistically, TAX1BP1-driven lysophagy requires its N-terminal SKICH domain, which binds both TBK1 and the autophagy regulatory factor RB1CC1, and requires upstream ubiquitylation events for efficient recruitment and lysophagic flux. These results identify TAX1BP1 as a central component in the lysophagy pathway and provide a proteomic resource for future studies of the lysophagy process.

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Phosphosite mapping on PI3Kc1 by ULK1 and TBK1 kinases.

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