Signal Amplification by an RTK/Abl Kinase Module

描述（由申请人提供）：本研究的目的是确定蛋白酪氨酸激酶和接头蛋白调节神经肌肉接头（NMJ）的机制，NMJ是神经肌肉接触部位的胆碱能突触。在NMJ适当的神经化学通讯需要高密度的突触后乙酰胆碱受体（AChR）的集群。当突触前神经分泌糖蛋白聚集蛋白时，这种聚集发生在发育的早期，糖蛋白聚集蛋白反过来激活肌肉特异性受体酪氨酸激酶（MUSK）。虽然这一事件已经作为突触诱导的范例超过十年，但关于将聚集蛋白诱导的MuSK激活与AChR聚集联系起来的信号网络知之甚少。我们最近确定了Abl家族的非受体酪氨酸激酶，Abl和精氨酸，作为关键介质的突触后组装下游聚集蛋白和MUSK。我们发现，Abl激酶定位于突触后膜的NMJ在体内和Abl激酶活性所需的聚集蛋白诱导AChR集群和增强MuSK酪氨酸磷酸化在肌管培养。此外，聚集蛋白刺激培养的肌管增加内源性Abl激酶活性，并诱导形成有助于相互酪氨酸磷酸化的MuSK/Abl复合物。基于我们的新发现，我们假设Abl家族激酶是NMJ形成和稳定所必需的，并为发育中的突触提供信号放大所需的酪氨酸激酶活性和组装和重塑所需的细胞骨架调节活性。为了验证这一假设，我们提出了以下具体目标：1）确定的功能性后果的依赖性MuSK磷酸化; 2）阐明的机制，Abl激酶调节聚集蛋白诱导的AChR集群; 3）确定额外的组件突触后MuSK/Abl信号复合物;和4）定义在小鼠NMJ的Abl家族激酶的体内作用。我们的研究结果将提供一个机制的理解受体和非受体酪氨酸激酶和衔接蛋白的形成和稳定的NMJ的作用。此外，他们将揭示新的光信号级联影响的疾病源于异常的NMJ功能，如先天性肌无力综合征和重症肌无力，并可能有广泛的影响，中央突触的形成和细胞间通讯，一般。

DESCRIPTION (provided by applicant): The goal of this research is to define the mechanisms by which protein tyrosine kinases and adaptor proteins regulate the neuromuscular junction (NMJ), a cholinergic synapse at sites of nerve-muscle contact. Proper neurochemical communication at the NMJ requires high density clustering of postsynaptic acetylcholine receptors (AChRs). This clustering occurs early in development when the presynaptic nerve secretes the glycoprotein agrin, which, in turn, activates the muscle-specific receptor tyrosine kinase (MUSK). Although this event has served as the paradigm of synapse induction for over a decade, little is known regarding the signaling network that links agrin-induced activation of MuSK to AChR clustering. We have recently identified the Abl family of nonreceptor tyrosine kinases, Abl and Arg, as critical mediators of postsynaptic assembly downstream of agrin and MUSK. We showed that Abl kinases localize to the postsynaptic membrane of the NMJ in vivo and that Abl kinase activity is required for agrin-induced AChR clustering and enhancement of MuSK tyrosine phosphorylation in myotube culture. Further, agrin stimulation of cultured myotubes increases endogenous Abl kinase activity and induces formation of a MuSK/Abl complex conducive to reciprocal tyrosine phosphorylation. Based on our novel findings, we hypothesize that Abl family kinases are required for the formation and stabilization of the NMJ and provide the developing synapse both the tyrosine kinase activity required for signal amplification and the cytoskeletal regulatory activity required for assembly and remodeling. To test this hypothesis, we propose the following specific aims: 1) determine the functional consequences of Abl-dependent MuSK phosphorylation; 2) elucidate the mechanisms by which Abl kinases regulate agrin-induced AChR clustering; 3) identify additional components of the postsynaptic MuSK/Abl signaling complex; and 4) define the in vivo role of Abl family kinases at the murine NMJ. Our results will provide a mechanistic understanding of the role of receptor and nonreceptor tyrosine kinases and adaptor proteins in the formation and stability of the NMJ. Moreover, they will shed new light on the signaling cascades affected in diseases stemming from aberrant NMJ function, such as the congenital myasthenic syndromes and myasthenia gravis, and may have broad implications for central synapse formation and intercellular communication, in general.