LRP6 phosphorylation in Wnt/beta-catenin signaling

描述(申请人提供)：WNT信号通过典型的a-连环蛋白途径控制细胞命运决定和细胞增殖，对动物发育是必不可少的。Wnt/b-catenin信号缺陷与人类结直肠癌、家族性骨质疏松症和其他疾病有关。因此，了解Wnt/b-catenin信号转导与人类健康密切相关。Wnt/b-catenin信号是由两个不同的细胞表面受体家族启动的。一个是蛇纹石受体FrizzledFZ家族的成员，另一个是低密度脂蛋白受体相关蛋白家族的单一跨膜受体，LRP5或LRP6。Wnt如何导致这些受体的激活是一个关键但知之甚少的问题。何博士的研究小组发现，FZ和LRP5/6在体外形成了Wnt诱导的共受体复合体，LRP6在信号转导过程中起着关键作用。他们最近发现，磷酸化可能是LRP6激活的基础。他们发现，在LRP5/6的胞内区重复5次的PPP(S/T)P基序对于LRP6的信号功能是必不可少的。他们证明了这个PPP(S/T)P基序的磷酸化是信号传递和Axin结合/识别所必需的，Axin是调节b-catenin稳定性的关键支架蛋白。他们进一步表明，Wnt在体内诱导LRP6磷酸化。这些结果表明，Wnt通过诱导LRP6的磷酸化来激活跨膜信号。在本申请中，设计了3个具体目标来充实/扩展这一工作模式。(1)对LRP6胞内区的5个PPP(S/T)P基序进行全序列分析。有待解决的问题包括这些PPP(S/T)P基序的功能以及它们的差异磷酸化是否控制LRP6信号转导。(2)研究轴蛋白与磷酸化PPP(S/T)P基序的相互作用。其目的是识别识别PPP(S/T)P磷酸化的Axin结构域/模块，并研究LRP6-Axin结合是否改变Axin复合体的组成，从而调节b-catenin的稳定性。(3)鉴定磷酸化PPP(S/T)P基序的一个或多个激酶。本研究的目的是研究Wnt/a-catenin信号转导通路在PPP(S/T)P磷酸化中的潜在作用，并通过几种互补的方法分离PPP(S/T)的一个或多个蛋白激酶。这些实验将显著提高对Wnt受体激活和Wnt信号转导在发育和疾病中的理解。

DESCRIPTION (provided by applicant): Wnt signaling through the canonical a-catenin pathway controls cell fate determination and cell proliferation, and is essential for animal development. Defective Wnt/b-catenin signaling has been associated with human colorectal cancer, familial osteoporosis, and other diseases. Thus understanding Wnt/b-catenin signaling is highly relevant to human health. Wnt/b-catenin signaling is initiated by 2 distinct families of cell surface receptors. One (1) is a member of the Frizzled (Fz) family of serpentine receptors, and the other is a single transmembrane receptor of the LDL receptor related protein family, LRP5 or LRP6. How Wnt leads to the activation of these receptors is a critical but poorly understood issue. Dr. He's group showed that Fz and LRP5/6 form a Wnt-inducible co-receptor complex in vitro, and that LRP6 plays a key role in the signaling process. They recently discovered that phosphorylation likely underlies LRP6 activation. They found that a PPP(S/T)P motif, which is reiterated 5 times in the intracellular domain of LRP5/6, is essential for LRP6 signaling function. They demonstrated that phosphorylation of this PPP(S/T)P motif is required for signaling and for binding/recognition by Axin, a key scaffolding protein that regulates b-catenin stability. They further showed that Wnt induces LRP6 phosphorylation in vivo. These results suggest that Wnt activates transmembrane signaling via inducing LRP6 phosphorylation. In this application, 3 specific aims are designed to substantiate/extend this working model. (1) To characterize fully all five PPP(S/T)P motifs in LRP6 intracellular domain. Issues to be addressed include the function of these PPP(S/T)P motifs and whether their differential phosphorylation controls LRP6 signaling. (2) To investigate Axin interaction with the phosphorylated PPP(S/T)P motifs. The aim is to identify the Axin domain/module that recognizes PPP(S/T)P phosphorylation, and examine whether LRP6-Axin association alters the composition of the Axin complex, thereby governing b-catenin stability. (3) To identify kinase (or kinases) that phosphorylates the PPP(S/T)P motif. This aim is to investigate known kinases implicated in Wnt/a-catenin signaling for their potential roles in PPP(S/T)P phosphorylation and to isolate the PPP(S/T)P kinase or kinases via several complementary approaches. These experiments will significantly enhance the understanding of Wnt receptor activation and Wnt signal transduction in development and disease.