siRNA screening of the kinome identifies ULK1 as a multidomain modulator of autophagy

Edmond Y W Chan, Serkan Kir, Sharon A Tooze

Research output: Contribution to journalArticle

294 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)25464-25474
Number of pages11
JournalJournal of Biological Chemistry
Volume282
Issue number35
DOIs
Publication statusPublished - 31 Aug 2007

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Keywords

  • amino acid motifs
  • amino acid sequence
  • animals
  • antibiotics, antineoplastic
  • autophagy
  • cytoplasm
  • HeLa cells
  • humans
  • intracellular signaling peptides and proteins
  • peptide mapping
  • protein kinases
  • protein structure, tertiary
  • protein transport
  • protein-serine-threonine kinases
  • RNA, small interfering
  • rats
  • sequence deletion
  • sirolimus
  • TOR serine-threonine kinases

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