siRNA screening of the kinome identifies ULK1 as a multidomain modulator of autophagy.
Autophagy is a vital response to nutrient starvation. Here, we screened a kinase-specific siRNA library using an autophagy assay in human embryonic kidney 293 cells that measures lipidation of the marker protein GFP-LC3 following amino acid starvation. This screen identified ULK1 in addition to other novel candidates that could be confirmed with multiple siRNAs. Knockdown of ULK1, but not the related kinase ULK2, inhibited the autophagic response. Also, ULK1 knockdown inhibited rapamycin-induced autophagy consistent with a role downstream of mTOR. Overexpression of ULK1 inhibited autophagy and this inhibition was independent of its kinase activity. Deletion of the PDZ domain-binding Val-Tyr-Ala motif at the ULK1 C terminus generated a more potent dominant-negative protein. Further deletions revealed that the minimal ULK1 dominant-negative region could be mapped to residues 1-351. Full-length ULK1 localized to cytoplasmic structures, some of which were GFP-LC3-positive, and this localization required the conserved C-terminal domain. In contrast, ULK1-(1-351) was diffuse in the cytoplasm. These experiments reveal at least two domains in ULK1 which likely function via unique sets of effectors to regulate autophagy.
Chan
Edmond Y W
EY
Secretory Pathways Laboratory, Cancer Research UK London Research Institute, London WC2A 3PX, United Kingdom.
Kir
Serkan
S
Tooze
Sharon A
SA
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D000071189:Autophagy-Related Protein-1 Homolog; D003593:Cytoplasm / Q000201:enzymology*; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017384:Sequence Deletion; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases
D000903:Antibiotics, Antineoplastic; D047908:Intracellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; D011494:Protein Kinases; C546842:MTOR protein, human; D000071189:Autophagy-Related Protein-1 Homolog; D017346:Protein Serine-Threonine Kinases; D058570:TOR Serine-Threonine Kinases; C484754:ULK1 protein, human; D020123:Sirolimus