Mapping the phosphorylation sites of Ulk1.
Ulk1 is a serine/threonine kinase that controls macroautophagy, an essential homeostatic recycling pathway that degrades bulk cytoplasmic material and directs the turnover of organelles such as peroxisomes and mitochondria. Further, macroautophagy is potently induced by signals that trigger metabolic stress, such as hypoxia and amino acid starvation, where its recycling functions provide macromolecules necessary to maintain catabolic metabolism and cell survival. Substrates for Ulk1 have not been identified, and little is known regarding post-translational control of Ulk1 kinase activity and function. To gain insights into the regulatory mechanisms of Ulk1, we developed a robust purification protocol for Ulk1 and demonstrated that Ulk1 is highly phosphorylated and requires autophosphorylation for stability. Importantly, high-resolution, tandem mass spectrometry identified multiple sites of phosphorylation on Ulk1, including several within domains known to regulate macroautophagy. Differential phosphorylation analyses also identified sites of phosphorylation in the C-terminal domain that depend upon or require Ulk1 autophosphorylation.
Dorsey
Frank C
FC
Department of Cancer Biology, The Scripps Research Institute, Jupiter, Florida 33458, USA. cdorsey@scripps.edu
Rose
Kristie L
KL
Coenen
Silvia
S
Prater
Stephanie M
SM
Cavett
Valerie
V
Cleveland
John L
JL
Caldwell-Busby
Jennifer
J
D000595:Amino Acid Sequence; D000818:Animals; D001343:Autophagy / Q000502:physiology; D000071189:Autophagy-Related Protein-1 Homolog; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011392:Proline / Q000737:chemistry; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods
D020033:Protein Isoforms; D012694:Serine; D011392:Proline; D000071189:Autophagy-Related Protein-1 Homolog; D017346:Protein Serine-Threonine Kinases; C112444:Ulk1 protein, mouse