siRNA Screening of the Kinome Identifies ULK1 as a Multidomain Modulator of Autophagy

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. Fulllength 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.During macroautophagy, a membrane cisterna wraps around cytoplasmic material to form a nascent autophagosome, which subsequently fuses with degradative endosomes. The targets of autophagy can include long-lived proteins, damaged organelles, ubiquitinated cellular substrates, and aberrant protein aggregates (1-4). Macroautophagy, which we refer to here as autophagy, is rapidly induced in response to amino acid deprivation and acts to recycle free molecular building blocks to the starving cell. Reflecting this cellular metabolic role, autophagy also appears to be critical in the maintenance of proper nutritional balance in the context of neonatal mice, developing Dictyostelium, and Caenorhabditis elegans undergoing development arrest (1, 5-7). Autophagy is also a major component of type-II non-apoptotic programmed cell death, and this may form part of the response to certain types of cell stress (8 -12). The recent implication of autophagy in medical contexts such as cancer, neurodegeneration and immunity have increased interest in elucidating the basic molecular mechanisms of the process (4, 13-16).Two yeast screens pioneered the identification of molecular components required for autophagy and a unified nomenclature has been devised for these genes (17)(18)(19). Although the function for many of these genes is unknown, a large proportion can be grouped into distinct biochemical pathways. Studies in yeast and mammalian cells have established the stepwise progression of these pathways, which appear to be essential at an early stage of autophagy (20, 21). For example, the gene products of Atg3, Atg4, Atg5, Atg7, Atg8, Atg10, Atg12, and Atg16 function coordinately in two interrelated ubiquitin-like conjugation systems (22-24). Importantly, yeast Atg8p and its mammalian homologs become covalently linked to a ph...

ULK-Atg13-FIP200 Complexes Mediate mTOR Signaling to the Autophagy Machinery

“…The ULK1 C-terminus is also involved in binding SynGAP and syntenin the proteins regulating the neuronal axon outgrowth, and is important for the autophagosomal localization of ULK1 (Tomoda et al, 2004;Chan et al, 2007). ULK1 also interacts with GATE-16 and GABARAP, the mammalian homologues of Atg8 that localize to autophagosome (Okazaki et al, 2000;Kabeya et al, 2004).…”

“…Atg17, which is also required for Atg1 activity, is specifically involved in autophagy but not in the Cvt pathway and is proposed to play a scaffold role (Suzuki et al, 2007). In mammals, two Atg1 homologues ULK1 and ULK2 have been identified and investigated (Chan et al, 2007;Hara et al, 2008;Kundu et al, 2008). Although overexpression of the kinase-dead mutants of ULK1 or ULK2 led to inhibition of autophagy, their roles in the regulation of autophagy have not been clearly defined.…”

Apoptosis and autophagy: regulatory connections between two supposedly different processes

“…The induction of autophagy occurs through a mechanism that involves the Atg1 kinase, which when overexpressed in Drosophila is sufficient to induce autophagy (31). In mammalian cells the Atg1 ortholog ULK1 also controls autophagy (32). Nucleation of the autophagic vesicle involves a Type III PI3 kinase complex that includes the kinase (Vps34) and Beclin1 (Atg6), which interact through a conserved domain in Beclin 1 (33).…”

A current perspective of autophagosome biogenesis

“…Upon starvation, decreased TORC1 activity results in dephosphorylation of Atg13, leading to the incorporation of Atg1-Atg13 into the Atg17-Atg29-Atg31 complex [23,24]. In mammals, the orthologues of Atg1 (ULK1 and 2; of the two, ULK1 is thought to play the major role in autophagy [25,26]) and Atg13 exist, but those of Atg17, Atg29, and Atg31 have not been identified. Instead, FIP200 and Atg101 are thought of as the functional counterparts of yeast Atg17-Atg29-Atg31.…”