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

描述（由申请方提供）：努南综合征（NS）是一种常染色体显性遗传疾病，发生率约为1：2，000活产婴儿。大约50%的NS患者在人PTPN 11基因中含有功能获得性突变，该基因编码含SH 2结构域的蛋白酪氨酸磷酸酶SHP-2。NS患者表现出多种多样的临床表现，最显著的是先天性心脏病（CHD）。高达80%的NS患者发生CHD，使得PTPN 11/SHP-2突变成为CHD最常见的非染色体原因。因此，改变的酪氨酰磷酸化是CHD的基础。这项研究的广泛目标是揭示NS相关的SHP-2突变如何引起CHD的分子基础。尽管大量的研究已经证实Ras/细胞外信号调节激酶1和2（ERK 1/2）通路的激活增强是NS介导的CHD的原因，但是NS相关的SHP-2突变参与Ras/ERK 1/2的病理生理信号传导的确切机制仍然未知。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ITIM的跨膜糖蛋白相互作用。我们推测，NS相关的SHP-2突变体引起的膜接近失调会引起Ras/ERK 1/2信号传导底物的混杂去磷酸化。将定义这些ITIM/NS-SHP-2相互作用对Ras/ERK 1/2信号的贡献。参与NS介导的Ras/ERK 1/2活性的底物将被鉴定并表征其参与NS相关的SHP-2突变体信号传导。在具体的目标二中，已经鉴定含有ITIM的跨膜糖蛋白在NS的小鼠模型中是高酪氨酰磷酸化的。我们将鉴定NS诱导的酪氨酸激酶，并使用遗传学和生物化学方法的组合来确定这种酪氨酸激酶是否传播增强的ERK 1/2激活和随后的NS相关的心脏缺陷。第三个目标将测试NS相关的SHP-2突变体作为NS介导的心脏缺陷的决定因素的改变的膜募集的病理生理学贡献。我们将通过采用遗传方法干扰NS相关的SHP-2向膜的募集来实现这一点。这些研究的完成将对CHD中NS相关SHP-2突变体的直接靶点产生新的见解，并可能揭示新的和已建立的信号分子在这种疾病中的意想不到的作用。冠心病靶点的确定也将揭示新的治疗策略模式，以及评估NS相关冠心病的预后工具。

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.