Signaling by gain-of-function SHP-2 mutants in Noonan syndrome

描述(申请人提供)：努南综合征(NS)是一种常染色体显性遗传性疾病，发生的频率约为1：2,000活产儿。大约50%的NS患者含有人类PTPN11基因的功能获得突变，该基因编码含有SH2结构域的蛋白酪氨酸磷酸酶SHP-2。NS患者表现出各种各样的临床表现，最显著的是先天性心脏病(CHD)。高达80%的NS患者会发生CHD，这使得PTPN11/SHP-2突变成为CHD最常见的非染色体原因。因此，酪氨酸磷酸化的改变是冠心病的基础。这项研究的广泛目标是揭示NS相关的SHP-2突变如何导致CHD的分子基础。虽然许多工作已经证实RAS/细胞外信号调节激酶1和2(ERK1/2)通路的增强激活是NS介导的CHD的原因，但NS相关的SHP-2突变参与RAS/ERK1/2的病理生理信号转导的确切机制仍不清楚。我们建议确定NS相关的SHP-2突变体的直接上游和下游靶点，并确定这些靶点是否参与NS介导的CHD的发展。在第一个目标中，我们已经确定了NS相关的SHP-2突变体优先与含有ITIM的跨膜糖蛋白相互作用。我们推测，NS相关的SHP-2突变体失调的膜邻近性参与了底物的错配去磷酸化，从而引发了RAS/ERK1/2信号。将定义这些ITIM/NS-SHP-2相互作用对RAS/ERK1/2信号的贡献。参与NS介导的RAS/ERK1/2活性的底物将被确定和表征，因为它们参与了NS相关的SHP-2突变信号。在特定目的二，含有跨膜糖蛋白的ITIM在NS小鼠模型中被鉴定为高酪氨酸磷酸化。我们将鉴定NS诱导的酪氨酸激酶(S)，并使用遗传和生化方法相结合的方法来确定该酪氨酸激酶(S)是否传播ERK1/2激活增强以及随后的NS相关心脏缺陷。第三个目的将测试NS相关的SHP-2突变体的膜改变募集作为NS介导的心脏缺陷的决定因素的病理生理学贡献。我们将通过使用遗传方法干扰NS相关的SHP-2到膜的募集来实现这一点。这些研究的完成将为NS相关的SHP-2突变在CHD中的直接靶点提供新的见解，并可能揭示新的和已建立的信号分子在这种疾病中意想不到的作用。识别与CHD相关的靶点还将揭示治疗NS相关CHD的新的治疗策略模式和评估预后的工具。

DESCRIPTION (provided by applicant): Noonan syndrome (NS) is an autosomal dominant disorder that occurs with a frequency of ~ 1:2,000 live births. Approximately 50% of NS patients contain a gain-of-function mutation in the human PTPN11 gene which encodes for the SH2 domain-containing protein tyrosine phosphatase, SHP-2. NS patients exhibit a diverse array of clinical manifestations, most notably, congenital heart disease (CHD). CHD occurs in up to 80% of NS patients, making PTPN11/SHP-2 mutations the most common non-chromosomal cause of CHD. Therefore, altered tyrosyl phosphorylation underlies the basis for CHD. The broad goal of this research is to uncover the molecular basis for how NS-associated SHP-2 mutations give rise to CHD. Although much work has established that enhanced activation of the Ras/extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway is causal to NS-mediated CHD the precise mechanisms through which NS-associated SHP-2 mutations engage in pathophysiological signaling to Ras/ERK1/2 remains unknown. We propose to identify the direct upstream and downstream targets of NS-associated SHP-2 mutants and determine if these targets are involved in the development of NS-mediated CHD. In the first aim, we have identified that NS-associated SHP- 2 mutants interact preferentially with ITIM-containing transmembrane glycoproteins. We hypothesize that dysregulated membrane proximity by NS-associated SHP-2 mutants engages promiscuous dephosphorylation of substrates that evoke Ras/ERK1/2 signaling. The contribution of these ITIM/NS-SHP-2 interactions to signal to Ras/ERK1/2 will be defined. The substrates involved in NS-mediated Ras/ERK1/2 activity will be identified and characterized for their involvement in NS-associated SHP-2 mutant signaling. In specific aim two, the ITIM containing transmembrane glycoproteins have been identified to be hypertyrosyl phosphorylated in a mouse model of NS. We will identify the NS-induced tyrosine kinase(s) and using a combination of genetic and biochemical approaches determine whether this tyrosine kinase(s) propagates enhanced ERK1/2 activation and subsequently NS-related cardiac defects. The third aim will test the pathophysiological contribution of altered membrane recruitment of NS-associated SHP-2 mutants as a determinant of NS-mediated cardiac defects. We will accomplish this by employing genetic approaches to interfere with the recruitment of NS- associated SHP-2 to the membrane. The completion of these studies will yield new insight into the direct targets of NS-associated SHP-2 mutants in CHD, and may reveal unanticipated roles, for new and established, signaling molecules in this disease. The identification of targets involved in CHD will also reveal new modes of therapeutic strategies in which to treat, and prognostic tools in which to evaluate, NS-related CHD.