The role of a CK1 kinase in membrane traffic

 描述（由申请人提供）：了解囊泡运输的方向性如何维持一直是膜运输领域的一个长期存在的问题。最近，我们已经表明，丝氨酸/苏氨酸激酶Hrr 25，酪蛋白激酶I（CKI）家族成员，有助于ER衍生的COPII包被囊泡的高尔基体的定向交付。这些研究揭示了蛋白磷酸化在有序囊泡靶向、囊泡脱壳和融合事件中的重要性的先前未被认识的作用。Hrr 25/CKId是在膜运输和其他细胞过程中起作用的丝氨酸/苏氨酸激酶家族的一个成员，该家族知之甚少，但高度保守。CKI家族成员被认为是组成型活性激酶，通过亚细胞定位和区室化调节激酶-底物相互作用。然而，最近，我们发现Rab GT3 Ypt 1（哺乳动物中的Rab 1）上调Hrr 25激酶活性。Rabs是一种分子开关，它在细胞膜上循环。它们被鸟嘌呤核苷酸交换因子（GEF）募集到膜上，GEF在特定位置激活Rab。Ypt 1调节Hrr 25活性的能力确保该激酶仅磷酸化COPII外壳的囊泡结合池。我们提出了四个具体的目标，以解决CKI激酶在膜交通的作用。这些研究可能作为其他CKI家族成员在膜运输中的作用的范例。1.我们将使用各种方法（共沉淀，体外磷酸化测定，差异分馏，柱层析，体外运输和uncoating测定），以探讨的可能性，Rab GTIPYpt 1调节uncoating的COPII囊泡通过其能力，以调节磷酸化的辅伴侣，这是一个Hrr 25底物。2.我们将讨论COPII和COPI囊泡是否通过共同机制未包衣。具体地说，我们将询问，如果相同的机器，去除COPII涂层囊泡也发挥了作用，在拆除高尔基体衍生囊泡的COPI笼。3.我们将询问另一CKI激酶家族成员Yck 3是否可以替代ER-高尔基体交通中的Hrr 25。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.