Signal Amplification by an RTK/Abl Kinase Module

描述(申请人提供)：这项研究的目标是确定蛋白酪氨酸激酶和适配器蛋白调节神经肌肉接头(NMJ)的机制，神经肌肉接头是神经-肌肉接触部位的胆碱能突触。NMJ的神经化学通讯需要突触后乙酰胆碱受体(AChRs)的高密度聚集。这种聚集性发生在发育早期，突触前神经分泌糖蛋白集聚蛋白，进而激活肌肉特异性受体酪氨酸激酶(Musk)。虽然这一事件作为突触诱导的范例已经有十多年了，但对于将集聚蛋白诱导的穆斯克激活与AChR聚集性联系起来的信号网络知之甚少。我们最近发现Abl家族的非受体酪氨酸激酶，Abl和Arg，是agrin和Musk下游突触后组装的关键介质。我们发现Abl激酶在体内定位于NMJ的突触后膜上，并且在肌管培养中，Abl激酶活性对于agrin诱导的AChR聚集和增强麝香的酪氨酸磷酸化是必需的。此外，对培养的肌管的集聚蛋白刺激增加了内源性Abl激酶的活性，并诱导形成有利于相互酪氨酸磷酸化的Musk/Abl复合体。根据我们的新发现，我们假设Abl家族的激酶是形成和稳定NMJ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以及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)确定依赖Abl的麝香磷酸化的功能后果；2)阐明Abl激酶调节集聚蛋白诱导的AChR的机制；3)确定突触后Musk/Abl信号复合体的其他成分；以及4)确定Abl家族激酶在小鼠NMJ中的体内作用。我们的结果将从机制上理解受体和非受体酪氨酸激酶和接头蛋白在NMJ的形成和稳定性中的作用。此外，它们还将揭示由NMJ功能异常引起的疾病中影响的信号级联，如先天性肌无力综合征和重症肌无力，并可能对中枢突触形成和细胞间通讯产生广泛的影响。

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.