PO-Mediated Signaling and Myelination

描述（由申请人提供）：髓磷脂是围绕中枢和外周神经系统P0中轴突的多层膜，是粘附分子免疫球蛋白家族的成员，是外周髓磷脂中最丰富的蛋白质，并且被认为在髓鞘缠绕轴突时介导多层髓鞘之间的粘附。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，以发现人类髓鞘形成中P0功能的分子基础。我们最近发现，点突变的蛋白激酶C（PKC）的目标基序- RSTK -在胞质结构域的P0废除其粘附功能，抑制PKC。我们还发现了一个CMT 1B患者的RSTK基序突变（R到S），进一步表明PKC介导的磷酸化在髓鞘形成中的重要性。该建议的目的是确定PKC介导的磷酸化在粘附和髓鞘形成中的作用。1)我们已经发现RACK 1，活化C激酶的受体是与P0的胞质结构域相关的蛋白质复合物的组分。我们将确定是否需要RACK 1或其他适配器将PKC靶向P0的胞质结构域。我们将进一步表征的结构域，通过它的合作伙伴相互作用，使用删除构建体结合双杂交系统和/或在体外结合试验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衔接子或效应子创建了一个结合位点。我们已经确定了一种蛋白质，其与P0的相互作用依赖于磷酸化的丝氨酸在RSTK基序使用酵母双杂交系统。我们将表征该组分和下游靶标的结合。3)我们将使用体外髓鞘形成培养系统，通过在与神经元共培养之前将显性阴性和组成型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物的构建体引入许旺细胞中，来评估目标1和2中鉴定的每个组分的作用。

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.