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J. Biol. Chem., Vol. 282, Issue 35, 25464-25474, August 31, 2007
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siRNA Screening of the Kinome Identifies ULK1 as a Multidomain Modulator of Autophagy*
From the Secretory Pathways Laboratory, Cancer Research UK London Research Institute, London WC2A 3PX, United Kingdom
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.
Received for publication, May 3, 2007 , and in revised form, June 25, 2007.
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and Figs. S1–S6.
1 To whom correspondence should be addressed: Secretory Pathways Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Field, London WC2A 3PX, UK. Tel.: 44-0-207-269-3045; E-mail: Sharon.Tooze{at}cancer.org.uk.
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