Mapping the Phosphorylation Sites of Ulk1
Mapping the Phosphorylation Sites of Ulk1
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DOI：
10.1021/pr900583m
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发表时间：
2009-11-01
影响因子：
4.4
通讯作者：
Caldwell-Busby, Jennifer
中科院分区：
文献类型：
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作者：
Dorsey, Frank C.;Rose, Kristie L.;Caldwell-Busby, Jennifer
Ulk1是一种丝氨酸/苏氨酸激酶，它控制宏观自噬，这是一种重要的体内平衡循环途径，降解大量的细胞质物质，并指导细胞器的周转，如过氧化物体和线粒体。此外，巨大的自噬是由触发代谢应激的信号有效地诱导的，例如缺氧和氨基酸饥饿，其循环功能提供维持分解代谢和细胞生存所需的大分子。Ulk1的底物尚未确定，关于翻译后对Ulk1活性和功能的调控也知之甚少。为了深入了解Ulk1的调控机制，我们开发了一种可靠的Ulk1纯化方案，并证明了Ulk1是高度磷酸化的，需要自身磷酸化才能保持稳定。重要的是，高分辨率串联质谱学确定了Ulk1上的多个磷酸化位点，包括已知的调控大自噬的结构域中的几个位点。差异磷酸化分析还确定了C-末端结构域中依赖或需要Ulk1自动磷酸化的磷酸化位点
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