Phosphorylation of LC3 by the Hippo Kinases STK3/STK4 Is Essential for Autophagy
Simon WilkinsonJinel S. JariwalaEricka L. AndersonKoyel MitraJill MeisenhelderJessica T. ChangTrey IdekerTony HunterVictor NizetAndrew DillinMalene Hansen
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Autophagy-related protein 13
Autophagosome
Autophagy is a process of regulated degradation. It eliminates damaged and unnecessary cellular components by engulfing them with a de novo‐ generated organelle: the double‐membrane autophagosome. The past three decades have provided us with a detailed parts list of the autophagy initiation machinery, have developed important insights into how these processes function and have identified regulatory proteins. It is now clear that autophagosome biogenesis requires the timely assembly of a complex machinery. However, it is unclear how a putative stable machine is assembled and disassembled and how the different parts cooperate to perform its overall function. Although they have long been somewhat enigmatic in their precise role, HORMA domain proteins (first identified in Hop1p, Rev7p and MAD2 proteins) autophagy‐related protein 13 (ATG13) and ATG101 of the ULK‐kinase complex have emerged as important coordinators of the autophagy‐initiating subcomplexes. Here, we will particularly focus on ATG13 and ATG101 and the role of their unusual metamorphosis in initiating autophagosome biogenesis. We will also explore how this metamorphosis could potentially be purposefully rate‐limiting and speculate on how it could regulate the spontaneous self‐assembly of the autophagy‐initiating machinery.
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Dephosphorylation
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Author(s): Lin, Mary Grace | Advisor(s): Hurley, James H | Abstract: Autophagy is an essential process in cells whereby, paradoxically, destruction of cellular components is necessary for cellular renewal, homeostasis, and survival under stresses ranging from starvation to infection. Substrates of autophagy, either bulk cytoplasmic contents or selectively targeted proteins and organelles, are engulfed in a growing double-membraned structure known as the phagophore. The phagophore matures and closes to form into the autophagosome, whose contents are degraded upon fusion of the autophagosome with the vacuole or lysosome. Autophagy occurs in all eukaryotes and many components of the autophagic machinery are conserved among yeast, animals, and plants. Many open questions remain as to how these individual components mechanistically work together to orchestrate autophagy, as well as how autophagy then functions in specific cellular contexts. Here, I study two protein complexes that are necessary for autophagy initiation, the Atg1 kinase complex and the phosphatidylinositol 3-kinase complex. In the first study, I use super-resolution, quantitative imaging of live cells coupled with structure-based mutational analysis to show that the Atg1 C-terminal domain has an essential function in autophagosome expansion that is downstream and separate from its Atg13-dependent role in autophagy initiation. The Atg1 C terminus is strikingly dynamic in the absence of Atg13, a phenomenon of unknown significance given that Atg1 and Atg13 have been thought to function in complex. The identification of an Atg13-independent role for the Atg1 suggests that these dynamics may be important for Atg1 function, particularly in autophagosome maturation. The second study examines the remarkable dynamics of the VPS34 kinase domain in context of the autophagic phosphatidylinositol 3-kinase complex. Using electron microscopy and both in vitro and in vivo assays of autophagy activity, we show that the dynamic dislodging of the VPS34 kinase domain is essential for autophagy initiation and that this movement is sterically inhibited by the VPS15 scaffold. Taking a step back from mechanistic intricacies and examining the ever-growing functions of autophagy in cellular context, in the last chapter I show that autophagy functions in organelle biogenesis. Specifically, autophagy degrades the protein OFD1 at centriolar satellites and promotes growth of the primary cilia, a sensory organelle. We identify a previously unknown substrate of autophagy and show that autophagic degradation of OFD1 can induce the formation of primary cilia in cancer cells, setting the stage for further investigations of the function of primary cilia and autophagy in cancer and other ciliopathies. Thus, this work spans studies of the molecular mechanisms of autophagy as well as its cellular functions.
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Autophagy is an intracellular degradative process with a number of roles, one of which can be the protection of eukaryotic cells from invading microbes. Microtubule-associated protein light-chain 3 (LC3) is a key autophagy-related protein that is recruited to the double-membrane autophagosome responsible for sequestering material intended for delivery to lysosomes. GFP-LC3 is widely used as a marker of autophagosome formation as denoted by the formation of green puncta when viewed by fluorescence microscopy. Recently, it has been demonstrated that LC3 can be recruited to other membranes including single-membrane phagosomes, in a process termed LC3-associated phagocytosis (LAP). Thus, the observation of green puncta in cells can no longer, by itself, be taken as evidence of autophagy. This review will clarify those features of LAP which serve to distinguish it from autophagy and that make connections with host autophagic responses in terms of infection by microbial pathogens. More specifically, it will refer to concurrent studies of the mechanism by which LAP is triggered in comparison to autophagy.
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Phosphorylation of proteins by protein kinases and dephosphorylation by protein phosphatases represents one of the most common, versatile, and perhaps confusing regulatory mechanisms in eukaryotic cells. Many, perhaps most, proteins in the eukaryotic nucleus are phosphoproteins, among them the proteins involved in DNA replication and its control. The importance of protein phosphorylation as a regulatory mechanism lies in its ready response to intracellular or extracellular signaling, its reversibility, and its ability to act as a measuring device to translate gradual changes into a molecular switch thrown at a threshold level of phosphorylation of a key target protein. The net level of protein phosphorylation is determined by the balance between the activities of protein kinases and those of protein phosphatases (for review, see Cohen 1989; Hubbard and Cohen 1993). The activities of kinases and phosphatases themselves are regulated by their own phosphorylation state. To complicate matters further, the number of known protein kinases, phosphatases, and regulatory subunits is growing rapidly, and their specificity is not always predictable from the primary sequence of the substrate protein (see Moreno and Nurse 1990; Cegielska et al. 1994a). Moreover, the effect of phosphorylation on the activity of a given target protein usually depends on the exact sites that are modified and unmodified. Thus, to understand how protein phosphorylation regulates the activity of replication proteins, one must know which sites in a target protein are phosphorylated, how physiological signals affect phosphorylation at each site, and how this phosphorylation influences the protein’s activity. Knowledge of protein kinases...
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Protein Kinases. Protein Phosphatases. Substrate Proteins. Direct Evidence for a Role of Protein Phosphorylation in Neuronal Function. Applications of Protein Phosphorylation Systems to Other Areas of Neuroscience. Initial Studies of the Molecular Biology of Protein Phosphorylation Systems. General Themes. Interactions Among Protein Phosphorylation Systems.
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