PO-Mediated Signaling and Myelination

描述(申请人提供)：髓鞘是一种多层膜，包围着中枢和周围神经系统的轴突P0，是黏附分子免疫球蛋白家族的成员，是周围髓鞘中含量最丰富的蛋白质，被认为在多层髓鞘包裹轴突时介导粘连。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都导致了人类周围神经病Charcot-Marie-Tooth病1B型。我们的长期目标是将基础研究和临床研究结合起来，以发现P0在人类髓鞘形成中作用的分子基础。我们最近发现，蛋白激酶C(PKC)靶标基序RSTK-在P0的细胞质结构域上的点突变会取消其黏附功能，抑制PKC也是如此。我们还鉴定了一例CMT1B患者的RSTK基序突变(R为S)，进一步表明了蛋白激酶C介导的磷酸化在髓鞘形成中的重要性。这项建议的目的是确定PKC介导的磷酸化在黏附和髓鞘形成中的作用。1)我们发现活化的C-激酶受体RACK1是与P0胞浆结构域相关的蛋白质复合体的一个组成部分。我们将确定是否需要RACK1或其他适配器将PKC靶向P0的细胞质结构域。我们将结合双杂交系统和/或体外结合试验2进一步表征伙伴相互作用的结构域。通过与其他黏附分子的类似，我们假设磷酸化为P0与下游靶点的相互作用创造了一个必不可少的接头或效应器的结合位点。我们使用酵母双杂交系统鉴定了一种蛋白质，其与P0的相互作用依赖于RSTK基序中丝氨酸的磷酸化。我们将表征该组件与下游目标的结合。3)我们将使用体外髓鞘培养系统评估AIMS 1和AIMS 2中确定的每个组分的作用，方法是在与神经元共同培养之前，将显性-负性和结构性活性结构，以及编码关键蛋白质-蛋白质相互作用的多肽竞争对手的结构引入雪旺细胞。

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.