During macroautophagy in mammalian cells, a membrane cisterna wraps around cytoplasmic material to form a nascent autophagosome, which then fuses with late endosomal structures to initiate the degradation of autophagosomal contents. The targets of macroautophagy (herein referred to as autophagy) can include long-lived proteins, organelles, ubiquitinated cellular substrates, and aberrant protein aggregates (7
). Autophagy has been implicated in a number of medical contexts, such as cancer, neurodegeneration, and immunity (as recently reviewed in references 16
, and 21
), raising interest in understanding its basic regulatory mechanisms.
The serine-threonine protein kinase Atg1 was originally identified as a critical autophagy regulator in genetic screens performed with the yeast Saccharomyces cerevisiae
). Autophagy in yeast is induced by nitrogen starvation or rapamycin treatment, and studies with yeast have shown that Atg1 functions at a regulatory step downstream of the nutrient-sensing signaling kinase TOR (target of rapamycin). Atg1 forms part of a complex that includes additional a
y (Atg) proteins, such as Atg13, Atg17, Atg29, Atg31, Atg11, Atg20, and Atg24 (9
). While the proteins Atg17, Atg29, and Atg31 have autophagy-specific functions, Atg11, Atg20, and Atg24 function in the yeast autophagy-related CVT (cytoplasm-to-vacuole targeting) pathway, raising the possibility that a number of different Atg1-containing subcomplexes may exist in vivo to regulate distinct pathways. Atg1 also regulates the recycling of Atg9 between the preautophagosomal structure (PAS) and a peripheral pool via a mechanism involving Atg2, Atg18, and the phosphatidylinositol-3 kinase complex I (25
). In addition, the localization of yeast Atg1 to the PAS can be regulated by cyclic AMP-dependent protein kinase-mediated phosphorylation in a starvation-dependent manner (2
Although the role of Atg1 kinase activity has been controversial (1
), recent data have demonstrated that Atg1 acts in distinct kinase-dependent and kinase-independent roles (5
). Kinase-inactivated Atg1 was capable of directing the assembly of the PAS containing Atg8, Atg17, and Atg29, consistent with the proposal that Atg1 plays a structural role. However, the kinase activity of Atg1 was required to drive protein or membrane dynamics through disassembly or dissociation of Atg proteins from the PAS, a step which is required to produce fully formed autophagosomes.
Full kinase activity of Atg1 in yeast requires its binding partners Atg13 and Atg17 (10
). Interestingly, the kinase activity of Atg1 is stimulated when autophagy is induced in yeast, and this correlates with increased binding of Atg1 to Atg13 and Atg17 upon autophagy induction (10
). Furthermore, the efficiency of Atg1-Atg13 binding is inversely correlated with levels of phosphorylation on Atg13 (10
), leading to the model in which Atg13 is dephosphorylated upon autophagy induction, thereby promoting its ability to bind to and act as a cofactor for Atg1. The autophagy-dependent kinases and phosphatases controlling yeast Atg13 phosphorylation remain to be determined. However, Atg13 dephosphorylation has been shown to be TOR dependent (10
The single Atg1 homologues of Dictyostelium discoideum, Caenorhabditis elegans
, and Drosophila melanogaster
have been confirmed to be key autophagy regulators (20
). In contrast, mammals have at least two Atg1 homologues (6
), Unc-51-like kinase 1 (ULK1) and ULK2, that share strong homology with the C. elegans
Atg1 homologue Unc (uncoordinated)-51 (32
). Whether ULK1 and ULK2 play similar roles for autophagy induction remains unclear.
We previously found that, in HEK293A cells, small interfering RNA (siRNA)-mediated knockdown of ULK1 was sufficient to reduce starvation-induced autophagy and inhibit the starvation-dependent redistribution of mammalian Atg9 (mAtg9) to a dispersed, peripherally localized pool (3
). In this cell system, knockdown of ULK2 had no effect on the induction of autophagy or mAtg9 traffic, suggesting a preferential role of ULK1 in autophagy. ULK1 and -2 have overlapping widespread mRNA expression patterns (35
). However, only ULK1 mRNA was upregulated in maturing reticulocyte cultures to promote autophagic clearance of mitochondria, indicating that some specificity exists in vivo (15
). These authors went on to show that mice lacking ULK1 displayed abnormal erythrocyte maturation but were viable and without a developmental phenotype, in contrast with other models of mice deficient in autophagy genes (13
). These data suggest that ULK2 can support autophagic function in the absence of ULK1, implying a more specialized role for ULK1 in vivo (15
). Although the precise roles of ULK1 and ULK2 require further clarification, our data on ULK1 and recent other data show that both of these proteins can localize to mammalian PASs (called isolation membranes, or phagophores) in a starvation-dependent manner (3
All Atg1 homologs share similar domain structures in which the kinase catalytic domain comprises the N-terminal one-third of the protein and the remaining two-thirds contain regulatory sequences. Comparison of the mouse ULK1 and ULK2 sequences with that of C. elegans
Unc-51 has allowed the definition of a C-terminal domain (CTD) (222 residues long in mouse ULK1) that shows relatively high levels of conservation (32
), suggestive of an important biological function. We previously described how the deletion of a three-residue PDZ binding motif at the CTD C terminus transforms ULK1 into a potent dominant inhibitor of autophagy (3
). In D. discoideum
, defects in development and autophagy found in Atg1 null cells could not be rescued with a mutant D. discoideum
Atg1 containing a deletion of the last 40 amino acids of the CTD (30
). A recent study has identified a novel ULK1-binding protein called FIP200 (focal adhesion kinase family-interacting protein of 200 kDa), which was required for starvation-induced autophagy, proper ULK1 phosphorylation, and ULK1 stability (6
). FIP200 binding to ULK1 required the CTD, further supporting a role of the CTD in autophagy regulation.
In this study, we aimed to gain insight into the function of ULK1 and ULK2 for the regulation of autophagy by studying the role of their kinase activities and the regulation of these activities by the CTD. We demonstrated that complete ablation of ULK1 kinase activity results in decreased protein autophosphorylation and a potent dominant-negative effect on autophagy. Our further analysis has led to a working model in which autophosphorylation is critical for promoting a closed molecular conformation that regulates interactions of the CTD. In further support of this model, expression of the CTD from either ULK1 or ULK2 was sufficient to inhibit autophagy, and additional analysis has identified a 7-residue motif critical for this effect. We determined that this motif was distinct from other signals within the CTD that direct membrane binding of ULK1 and ULK2 and incorporation into large protein complexes.
To explore other mechanisms involved in the function of the CTD, we identified a putative human Atg13 orthologue that we confirmed is essential for autophagy by using siRNA depletion. In addition, we found that loss of human Atg13 affected the trafficking of mAtg9, as was previously observed after the loss of ULK1 (37
). Although mAtg13 bound the CTDs of both ULK1 and ULK2, this interaction utilized sequences that were distinct from the dominant-negative 7-residue motif, and overexpression of Atg13 did not rescue the dominant-negative activity of the overexpressed CTDs. Thus, our data have identified a 7-residue motif in the ULK1 and ULK2 CTDs that engages a novel dominant-negative mechanism which is independent of Atg13 and membrane-associated components.