Autophagy is an evolutionarily conserved, degradative pathway that has been implicated
in a number of physiological processes such as development and aging as well as cancer
and innate immunity. This pathway is important for cell survival in starvation and is
considered as a potential target for therapeutic intervention in a number of pathological
conditions. Therefore, it is important that we develop a thorough understanding of the
mechanisms regulating this trafficking pathway. Autophagy was initially identified as a
cellular response to nutrient deprivation and is essential for cell survival during periods of
starvation. During autophagy, an isolation membrane emanates from a nucleation site that
is known as the phagophore assembly site (PAS). This membrane encapsulates nearby
cytoplasm to form an autophagosome that is ultimately targeted to the vacuole/lysosome
for degradation. The small molecules produced are then recycled and used by cells during
this period of starvation. Autophagy activity is highly regulated and multiple signaling
pathways are known to target a complex of proteins that contains the Atg1 protein kinase.
Atg1 protein kinase activity is essential for normal autophagy in all eukaryotes
and appears to be controlled tightly by a number of kinases, which target this enzyme and
its associated protein partners. Our data and that of others have established that Atg1
activity is regulated, at least in part, by protein phosphorylation. In this work, we
identified a particular phosphorylation event on Atg1 as an important control point within the autophagy pathway in Saccharomyces cerevisiae. This phosphorylation occurs at a
threonine residue, T226, within the Atg1 activation loop that is conserved in all Atg1
orthologs. This activation loop phosphorylation is essential for Atg1 kinase activity and
the induction of autophagy. The data also suggested that promoting this
autophosphorylation is a primary role for two key conserved regulators of Atg1 activity,
Atg13 and Atg17. Atg13, in particular, appears to stimulate this phosphorylation by
promoting an Atg1 self-interaction. In all, these data suggest that autophosphorylation
within the Atg1 activation loop may represent a point of regulatory control for this
degradative process.
To further our knowledge of phosphorylation in Atg1, we used a combined mass
spectrometry and molecular biology approach to identify and characterize additional sites
of phosphorylation in Atg1. Fifteen sites of phosphorylation were discovered here,
including nine that had not been noted previously. Alterations of these sites identified a
number of positions that appear to be important for full autophagy activity in vivo. One
site was of special interest as it was within a highly conserved motif, the Gly-rich loop, in
the Atg1 kinase domain. Phosphorylation at this equivalent position inhibits the kinase
activity of particular cyclin-dependent kinases and we showed here that this site may
serve a similar function in Atg1. In addition, we identified Ser-390 as the site of
autophosphorylation responsible for the anomalous migration observed for Atg1 on SDSpolyacrylamide
gels. In all, the analyses here identified a number of potential sites of
regulation in the Atg1 protein that provide important insight into the control of the
autophagy process and will form a framework for future studies with this enzyme.