Comparative Phosphoproteomics to Identify Targets of the Clock-Relevant Casein Kinase 1 in C. reinhardtii Flagella
Comparative Phosphoproteomics to Identify Targets of the Clock-Relevant Casein Kinase 1 in C. reinhardtii Flagella
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DOI：
10.1007/978-1-4939-0700-7_12
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发表时间：
2014-01-01
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影响因子：
--
通讯作者：
Mittag, Maria
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文献类型：
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作者：
Boesger, Jens;Wagner, Volker;Mittag, Maria
在绿色双鞭毛藻莱茵衣藻（Chlamydomonas reinhardtii）中，已经确定了不同的时钟相关成分，这些成分参与维持昼夜节律的相位、周期和幅度。很明显，其中一些与鞭毛功能相关，如CASEIN KINASE1 （CK1）。CK1参与维持周期。但它也与鞭毛的形成有关，鞭毛的物理位置，它控制着游泳的速度。在本章中，我们描述(1)鞭毛亚蛋白质组如何纯化，(2)如何富集来自该细胞器的磷酸肽，(3)如何确定体内磷酸化位点，以及(4)如何使用特定抑制剂找到CK1的直接和间接鞭毛靶标。如果有特定的抑制剂或突变体，这种方法也可以用于其他与时钟相关的激酶。
In the green biflagellate alga Chlamydomonas reinhardtii different clock-relevant components have been identified that are involved in maintaining phase, period, and amplitude of circadian rhythms. It became evident that several of them are interconnected to flagellar function such as CASEIN KINASE1 (CK1). CK1 is involved in keeping the period. But it is also relevant for the formation of flagella, where it is physically located, and it controls the swimming velocity. In this chapter, we describe (1) how the flagellar sub-proteome is purified, (2) how phosphopeptides from this organelle are enriched, (3) how in vivo phosphorylation sites are determined, and (4) how direct and indirect flagellar targets of CK1 can be found using a specific inhibitor. Such a procedure can also be employed with other clock-relevant kinases if specific inhibitors or mutants are available.