The role of a CK1 kinase in membrane traffic

 描述(由申请人提供)：了解如何维持囊泡运输的方向性一直是膜运输领域的一个长期问题。最近，我们发现酪蛋白激酶I(CKI)家族成员丝氨酸/苏氨酸激酶Hrr25参与了内质网COPII包被囊泡向高尔基体的定向传递。这些研究揭示了蛋白质磷酸化在排列小泡靶向、小泡去涂层和融合事件中的重要性，这是以前没有人认识到的作用。Hrr25/CKiD是一个知之甚少但高度保守的丝氨酸/苏氨酸激酶家族的成员，该家族在膜运输和其他细胞过程中发挥作用。CKI家族成员被认为是通过亚细胞定位和分区来调节激酶-底物相互作用的结构性活性激酶。然而，最近我们发现Rab GTP酶Ypt1(哺乳动物中的Rab1)上调Hrr25激酶的活性。RABS是一种分子开关，可以在膜上和膜上循环。它们被鸟嘌呤核苷酸交换因子(GEF)招募到膜上，激活特定位置的RAB。Ypt1调节Hrr25活性的能力确保了该激酶只会磷酸化COPII外壳的囊泡结合池。我们提出了四个特定的目标，以解决CKI激酶在膜交通中的作用。这些研究很可能成为研究其他CKI家族成员在膜运输中的作用的范例。1.我们将使用多种方法(共沉淀、体外磷酸化分析、差异分级、柱层析、体外转运和去涂层分析)来探讨Rab GTPase Ypt1通过调节作为Hrr25底物的辅伴侣的磷酸化来调节COPII囊泡去涂层的可能性。2.我们将讨论COPII和COPI囊泡是否没有被共同的机制包裹。具体地说，我们会问，剥离COPII涂层囊泡的相同机械是否也在将COPI笼子从高尔基体衍生的囊泡中拆解出来的过程中发挥作用。3.我们会问，在ER-Golgi流量中，另一个CKI激酶家族成员Yck 3是否可以取代Hrr25。4.我们将讨论其他CKI激酶家族成员在膜运输中的作用。

 DESCRIPTION (provided by applicant): Understanding how the directionality of vesicle traffic is maintained has been a long standing question in the field of membrane traffic. Recently, we have shown that the serine/threonine kinase Hrr25, a casein kinase I (CKI) family member, contributes to the directional delivery of ER-derived COPII coated vesicles to the Golgi. These studies have uncovered a previously unappreciated role for the importance of protein phosphorylation in ordering vesicle targeting, vesicle uncoating and fusion events. Hrr25/CKId is a member of a poorly understood, yet highly conserved, family of serine/threonine kinases that function in membrane traffic and other cellular processes. CKI family members were thought to be constitutively active kinases that regulate kinase-substrate interactions through subcellular localization and compartmentalization. Recently, however, we have found that the Rab GTPase Ypt1 (Rab1 in mammals) upregulates Hrr25 kinase activity. Rabs are molecular switches that cycle on and off membranes. They are recruited to membranes by guanine nucleotide exchange factors (GEFs) that activate the Rab at a specific location. The ability of Ypt1 to regulate the activity of Hrr25 ensures this kinase will only phosphorylate the vesicle-bound pool of the COPII coat. We propose four specific aims to address the role of CKI kinases in membrane traffic. These studies are likely to serve as a paradigm for the role of other CKI family members in membrane traffic. 1. We will use a variety of approaches (co-precipitation, in vitro phosphorylation assays, differential fractionation, column chromatography, in vitro transport and uncoating assays) to explore the possibility that the Rab GTPase Ypt1 regulates the uncoating of COPII vesicles via its ability to modulate the phosphorylation of a co-chaperone that is an Hrr25 substrate. 2. We will address if COPII and COPI vesicles are uncoated by a common mechanism. Specifically, we will ask if the same machinery that uncoats COPII coated vesicles also plays a role in disassembling the COPI cage from Golgi-derived vesicles. 3. We will ask if another CKI kinase family member, Yck3, can substitute for Hrr25 in ER-Golgi traffic. 4. We will address the role of other CKI kinase family members in membrane traffic.