Cell polarity and phosphoinositide kinases in Wnt signalling

在人类中，很大一部分结肠癌和很大一部分其他癌症是由于一种被称为“WNT”蛋白的化学信号的问题所致。正常情况下，WNT由体内的某些细胞发送到其他细胞，以促进健康的生长和更新。当这些信号变得过多时，就会导致癌症。Wnt信号不仅在癌症中很重要，在胚胎发育过程中的正常组织中也非常重要。在正确的时间和地点，WNT触发不同类型组织的形成和生长，从而使身体形成正确的形式。如果我们能够了解Wnt信号是如何工作的，我们可能会改进癌症治疗，并利用正常的胚胎过程来治愈和修复受伤害、疾病和衰老影响的组织。WNT信号的一个复杂之处在于，WNT可以触发三种不同的反应，目前还不清楚是什么决定了细胞将给出哪一种反应。我们最近发现了一种名为PI4KIIb的酶，当从青蛙胚胎中耗尽这种酶时，会导致产生的蝌蚪拥有更短、更宽的身体和更小的头部。(我们使用青蛙胚胎是因为它们可以大量获得，而且它们和它们的细胞很大，所以很容易操作和观察。)我们看到的更短、更宽的身体暗示着对所谓的非规范WNT信号的影响(有两种类型)。较小的头部暗示了典型的Wnt-β-catenin信号的增加的影响。有可能PI4KIIb对两者都有影响，但一种非常相似的酶PI4KIIa只影响典型信号，而从胚胎中去除后者会增加典型Wnt信号和头部大小。我们的第一个目标是直接测试我们看到的对蝌蚪外表的影响是否真的是由于不同的WNT路径上的影响，或者是否有其他事情正在发生。我们将测量在PI4KIIa和b增加或减少的蝌蚪中发生了多少每种反应。在以前的项目中，我们发现不同的Wnt途径分支受到另一种名为PAR-1的酶的不同版本的调节。我们现在已经发现PAR-1作用于PI4KIIb，在PI4KIIb上的特定位置添加一个磷酸基团。此外，我们发现PI4KIIb中的这个位点是它移动到细胞表面膜所必需的。其他研究表明，膜结合对PI4KIIb的活性至关重要。我们将测试PAR-1a是否以与PI4KIIb相同的方式影响PI4KIIa，方法是类似地突变靶点，并观察突变的蛋白是否被阻止进入细胞膜。我们还将测试耗尽不同版本的PAR-1是否会影响PI4KIIa和-b在胚胎中的定位，因为不同的PAR-1版本作用于不同的PI4KII版本。在建立了PI4KII途径活动和定位决定因素后，我们将看看PI4KIIb的这两个关键属性是否相关：PI4KII定位是否影响其在Wnt信号传递中的活性？我们将对PI4KII版本和突变体进行相同的途径分析，看看活性和定位是否相关。由于PAR-1总是在上皮性(层状)组织的基底部发现，我们将测试这样的想法，即PAR-1/PI4KII系统控制着当WNT被呈现到这些组织的不同侧时发生的细胞反应。这将是对不同途径的意义及其受PAR-1调控的重大洞察，并表明在癌症治疗或再生医学方法中，重要的不仅是是否应用模仿或抑制WNTS的细胞极性试剂，而且还涉及到在哪里应用。

A large proportion of colon cancers and a significant fraction of other cancers in humans are due to problems with a chemical signal known as the "wnt" protein. Wnt is sent by certain cells in the body to other cells normally to promote healthy growth and renewal. When these signals become excessive, cancer is the result. The Wnt signal, as well as being important in cancer, is also very important in normal tissues during embryo development. In the right places and times, Wnt triggers the formation of different kinds of tissues and their growth so that the body forms correctly. If we can understand how Wnt signalling works, we may improve cancer therapies and harness normal embryonic processes to heal and repair tissues affected by injury, disease and ageing. One complication of Wnt signalling is that Wnt can trigger three different kinds of response and it is not understood what determines which of these responses a cell will give. We recently found an enzyme, known as PI4KIIb, which, when depleted from frog embryos, causes the resulting tadpoles to have shorter, wider bodies and smaller heads. (We use frog embryos because they are available in large numbers and they and their cells are large and so easy to manipulate and observe.) The shorter, wider bodies we see are suggestive of effects on so-called non-canonical Wnt signalling (of which there are two types). The smaller heads are suggestive of effects of increased canonical Wnt-beta-catenin signalling. It is possible that PI4KIIb affects both, but a very similar enzyme, PI4KIIa, affects only canonical signalling, and depleting the latter from embryos increases canonical Wnt signalling and head size. Our first objective is to test directly whether the effects we see on the tadpole appearance really are due to the effects on the different Wnt pathways, or whether something else is going on. We will measure how much of each response occurs in tadpoles that have increased or decreased amounts of PI4KIIa and b. In a previous project, we found that the different Wnt pathway branches are regulated by different versions of another enzyme known as PAR-1. We have now found that PAR-1 acts on PI4KIIb, adding a phosphate group to a specific site on the latter. Moreover, we find that this site in PI4KIIb is needed for it to move to the cell's surface membrane. Others have shown that membrane association is critical for activity of PI4KIIb. We will test whether PAR-1a affects PI4KIIa in the same way as it does PI4KIIb by similarly mutating the target site and seeing whether the mutated protein is prevented from going to the cell membrane. We shall also test whether depleting different versions of PAR-1 affects the localisation of PI4KIIa and -b in embryos, with the idea that the different PAR-1 versions act on different PI4KII versions.Having established PI4KII pathway activities and localisation determinants, we shall see whether these two key properties of PI4KIIb are related: does PI4KII localisation affect its activity in Wnt signalling? We will do the same sets of pathway assays on the PI4KII versions and mutants and see whether the activity and localisation are correlated. Since PAR-1 is always found on the basal side of epithelial (layered) tissues, we will test the idea that the PAR-1/PI4KII system governs which cell responses occur when Wnt is presented to different sides of such tissues. This would be a major insight into the significance of the different pathways and their regulation by PAR-1 and show that in cancer therapy or regenerative medical approaches, it matters not just whether but also where in relation to the cells' polarity agents that mimic or inhibit Wnts are applied.