Activation of Atg1 kinase in autophagy by regulated phosphorylation.
Autophagy is a highly regulated trafficking pathway that leads to selective degradation of cellular constituents such as protein aggregates and excessive and damaged organelles. Atg1 is an essential part of the core autophagic machinery, which triggers induction of autophagy and the Cvt pathway. Although changes in Atg1 phosphorylation and complex formation are thought to regulate its function, the mechanism of Atg1 kinase activation remains unclear. Using a quantitative mass spectrometry approach, we identified 29 phosphorylation sites, of which five are either upregulated or downregulated by rapamycin treatment. Two phosphorylation sites, threonine 226 and serine 230, are evolutionarily conserved and located in the activation loop of the amino terminal kinase domain of Atg1. These phosphorylation events are not required for Atg1 localization to the phagosome assembly site (PAS), or the proper assembly of the multisubunit Atg1 kinase complex and binding to its activator Atg13. However, mutation of either one of these sites results in a loss of Atg1 kinase activity and its function in autophagy and the Cvt pathway. Taken together, our data suggest that phosphorylation of Atg1 on multiple sites provides critical mechanisms to regulate Atg1 function in autophagy and the Cvt pathway.
Kijanska
Monika
M
Institute of Biochemistry, Department of Biology, ETH Züric, Zürich, Switzerland.
Dohnal
Ilse
I
Reiter
Wolfgang
W
Kaspar
Susanne
S
Stoffel
Ingrid
I
Ammerer
Gustav
G
Kraft
Claudine
C
Peter
Matthias
M
D000595:Amino Acid Sequence; D001343:Autophagy / Q000187:drug effects; D000071183:Autophagy-Related Proteins; D017124:Conserved Sequence / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology
D000071183:Autophagy-Related Proteins; D029701:Saccharomyces cerevisiae Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D011494:Protein Kinases; C477823:ATG1 protein, S cerevisiae; D020123:Sirolimus