PO-Mediated Signaling and Myelination

描述（申请人提供）：髓磷脂是中枢和外周神经系统中包裹轴突的多层膜，是免疫球蛋白粘附分子家族的成员，是外周髓磷脂中最丰富的蛋白质，被认为在髓磷脂包裹轴突时介导多层髓磷脂之间的粘附。与P0在髓鞘形成中的重要作用一致，细胞外和细胞内结构域的突变导致人类周围神经病变1B型Charcot-Marie-Tooth病。我们的长期目标是结合基础和临床研究，以发现P0在人类髓鞘形成中功能的分子基础。我们最近发现，蛋白激酶C （PKC）靶基- RSTK -在P0细胞质结构域中的点突变破坏了它的粘附功能，PKC的抑制也是如此。我们还发现了一个RSTK基序（R到S）突变的CMT1B患者，进一步表明pkc介导的磷酸化在髓鞘形成中的重要性。这项提议的目的是确定pkc介导的磷酸化在粘连和髓鞘形成中的作用。1)我们发现活化C激酶受体RACK1是与P0细胞质结构域相关的蛋白复合物的一个组成部分。我们将确定是否需要RACK1或其他适配器将PKC靶向到P0的细胞质结构域。我们将使用删除构建体结合双杂交系统和/或体外结合试验进一步表征合作伙伴相互作用的结构域2。通过与其他粘附分子的类比，我们假设磷酸化为P0与下游靶标相互作用所必需的接头或效应物创造了一个结合位点。我们利用酵母双杂交系统鉴定了一种与P0相互作用依赖于RSTK基序丝氨酸磷酸化的蛋白。我们将描述该组分与下游靶标的结合。3)我们将在与神经元共培养之前，利用体外髓鞘培养系统，通过引入显性阴性和构成活性构建体，以及编码关键蛋白质-蛋白质相互作用的肽竞争体的构建体，来评估目标1和目标2中确定的每个成分的作用。

DESCRIPTION (provided by applicant): Myelin is a multilamellar membrane that surrounds axons in both the central and peripheral nervous system P0, a member of the immunoglobulin family of adhesion molecules, is the most abundant protein in peripheral myelin and is thought to mediate adhesion between the multiple layers of myelin as they wrap around the axon. Consistent with an important role for P0 in myelination, mutations in both the extracellular and intracellular domains cause the human peripheral neuropathy Charcot-Marie-Tooth disease type 1B. Our long-range goals are to couple basic and clinical research to discover the molecular basis for P0 function in human myelination. We have recently discovered that point mutations in a Protein Kinase C (PKC) target motif - RSTK - in the cytoplasmic domain of P0 abolish its adhesive function, as does inhibition of PKC . We have also identified a CMT1B patient with a mutation in the RSTK motif (R to S), further indicating the importance of PKC-mediated phosphorylation in myelination. The goals of this proposal are to determine the role of PKC-mediated phosphorylation in adhesion and myelination. 1) We have found that RACK1, the Receptor for Activated C Kinase is a component of the complex of proteins associated with the cytoplasmic domain of P0. We will determine if RACK1 or other adapters are needed to target PKC to the cytoplasmic domain of P0. We will further characterize the domains through which partners interact using deletion constructs in conjunction with the two-hybrid system and/or an in vitro binding assay 2. By analogy with other adhesion molecules, we hypothesize that phosphorylation creates a binding site for adaptors or effectors essential for the interaction of P0 with downstream targets. We have identified one protein whose interaction with P0 depends on phosphorylation of serine in the RSTK motif using the yeast two-hybrid system. We will characterize the binding of this component and downstream targets. 3) We will evaluate the role of each of the components identified in aims 1 and 2 using an in vitro myelination culture system by introducing dominant-negative and constitutively active constructs, as well as constructs coding for peptide competitors for critical protein-protein interactions, into Schwann cells prior to co-culture with neurons.