Cell polarity and phosphoinositide kinases in Wnt signalling

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.

One of the most important pathways in biology is the Wnt pathway because it regulates growth, proliferation and morphogenesis in normal development, repair and disease. The Wnt protein can elicit at least three types of response in cells, activating so-called Wnt-canonical, Wnt-PCP and Wnt-Ca branches. What determines which output is produced by a given cell is not well understood but is critical for how Wnts function in vivo. We previously showed that different branches of the Wnt pathway are regulated by different isoforms of a conserved polarity protein PAR-1, suggesting that the apical and basolateral domains of epithelial cells into which PAR-1 partitions cells may somehow have different Wnt responses. To define the mechanism of action of PAR-1, we recently identified a novel molecular substrate of PAR-1's kinase activity, namely phosphoinositol-4-kinase IIbeta (PI4KIIb). The PAR-1 phosphorylation site on PI4KIIb is required for its plasma membrane localisation. Depletion of PI4KIIb in Xenopus embryos suggests that PI4KIIb controls Wnt signalling in vivo and gives a phenotype very different from when PI4KIIa, the alpha isoform, is depleted. We propose to (1) measure and compare the effects and requirement of PI4KII isoforms on the different Wnt branches, (2) characterise the localisation of PI4KII isoforms and its regulation by PAR-1 isoforms, and (3) determine how PI4KII localisation affects different Wnt signal branch outputs. We will do this in Xenopus embryos because microinjection of mRNAs and reporter DNA provide rapid assays for the questions we are asking and the large cells of Xenopus facilitate localisation assays in a physiological context. In parallel we will test directly whether apical versus basal presentation of Wnts to epithelial cells determines which pathway is stimulated. Together these findings will open up novel interactions between cell polarity and Wnt signalling and provide insights into transduction of this key pathway.

Who will benefit from this research?
* Pharmaceutical companies need to know about drug delivery

* Biotechnology companies interested in regeneration of complex tissues

* The wider public including anyone who has an interest in biology

* Staff working on the project and students studying at this institution

How will they benefit from this research?

This proposal is for basic rather than directly applied research. Nonetheless, pharmaceutical companies strive to understand the pharmacology of their products and the directional basis of signalling specificity, which is what this proposal aims to elucidate, is significant for this understanding. Targetted cancer therapy, particularly of solid tumours, is in its infancy and survival rates are low. New ideas are needed in this field and taking cell polarity into account may be one such idea.
Biotechnology companies working on the directed differentiation of stem cells for therapeutic applications also need to know how their repertoire of differentiative agents is affected by the direction of delivery and the architecture of the cells being treated.
The wider public is interested in fundamental biology and does appreciate the beauty and architecture of the body and how it is formed.
Staff working on the project will acquire skills in embryology, molecular biology, imaging, project planning and management and science writing.

The research has the potential to contribute to the nation's health, wealth and culture in the same way that fundamental research on the action of morphogen growth factors explored by the PI in the early 1990s is now a key formative idea that drives and underpins stem cell therapies, including human embryonic stem cell clinical trials currently underway in the U.K. The wealth comes from maintaining a competitive biotechnology and pharmaceutical sector, the health from new therapies and the culture from outreach by the PI and other scientists in academia to educate and inform the wider public about the wonders of biology.