Whole-cell lysate was centrifuged for 10 min at 4° and the supernatant used for immunoprecipitation via Glutathione Agarose (Thermo Fisher Scientific, 16102), Streptavidin Beads (genscript, L00353) or anti-Flag M2 Affinity Gel (Sigma, A2220), followed by incubation at 4 °C overnight.
The Unc-51 like autophagy activating kinase 1 (ULK1) complex plays a central role in the initiation stage of autophagy. However, the function of ULK1 in the late stage of autophagy is unknown. Here, we report that ULK1, a central kinase of the ULK1 complex involved in autophagy initiation, promotes autophagosome-lysosome fusion. PKCα phosphorylates ULK1 and prevents autolysosome formation. PKCα phosphorylation of ULK1 does not change its kinase activity; however, it decreases autophagosome-lysosome fusion by reducing the affinity of ULK1 for syntaxin 17 (STX17). Unphosphorylated ULK1 recruited STX17 and increased STX17's affinity towards synaptosomal-associated protein 29 (SNAP29). Additionally, phosphorylati... More
The Unc-51 like autophagy activating kinase 1 (ULK1) complex plays a central role in the initiation stage of autophagy. However, the function of ULK1 in the late stage of autophagy is unknown. Here, we report that ULK1, a central kinase of the ULK1 complex involved in autophagy initiation, promotes autophagosome-lysosome fusion. PKCα phosphorylates ULK1 and prevents autolysosome formation. PKCα phosphorylation of ULK1 does not change its kinase activity; however, it decreases autophagosome-lysosome fusion by reducing the affinity of ULK1 for syntaxin 17 (STX17). Unphosphorylated ULK1 recruited STX17 and increased STX17's affinity towards synaptosomal-associated protein 29 (SNAP29). Additionally, phosphorylation of ULK1 enhances its interaction with heat shock cognate 70 kDa protein (HSC70) and increases its degradation through chaperone-mediated autophagy (CMA). Our study unearths a key mechanism underlying autolysosome formation, a process in which the kinase activity of PKCα plays an instrumental role, and reveals the significance of the mutual regulation of macroautophagy and CMA in maintaining the balance of autophagy.