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Physiological properties and functions of phosphatidylinositol 5-phosphate 4-kinase gamma

Reference	BB/J01575X/1
Principal Investigator / Supervisor	Professor Robin Irvine
Co-Investigators / Co-Supervisors
Institution	University of Cambridge
Department	Pharmacology
Funding type	Research
Value (£)	362,693
Status	Completed
Type	Research Grant
Start date	05/11/2012
End date	04/11/2015
Duration	36 months

Abstract

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are enigmatic inositol lipid kinases whose function is currently believed to be to regulate the cellular levels of their substrate, PI5P, an inositol lipid found at low levels in the cytoplasm and the nucleus of animal cells, and whose function has so far been associated with stress-activated signalling pathways. We have, primarily by the novel technique of genomic tagging in DT40 cells, made a series of discoveries about the three mammalian isoform, PI5P4Ks alpha, beta and gamma that throw a new light on their functional interrelationship. The alpha and beta isoforms form stable heterodimers in vivo randomly (that is, they dimerise in proportion to their relative concentrations), and we have now shown that PI5P4Ks alpha and gamma also heterodimerise in vitro. Moreover, we have discovered that the PI5P4Ks have very different enzymatic activities, with alpha being much more active than beta, which is in turn much more active than gamma. Our knowledge about PI5P4K gamma is at a very early stage, almost all of what is know being discovered in our lab, and this proposal is a focused, but methodologically wide-ranging, assault on this under-investigated enzyme. We have a knock-out mouse of PI5P4K gamma in the final stages of derivation, and in addition to these mice being monitored for functions likely to be compromised (kidney and neuronal function) by specialist collaborators, they will act as a source of tissues and cells for us to use ourselves to answer basic questions about precise cellular localisation and then function of PI5P4K gamma. These studies will be complemented by use of kidney cell lines (e.g. raTAL), and of Nalm-6 cells, the human equivalent of the chicken DT40s. We will knock out the PI5P4K gamma gene, or tag it suitably for localisation or rapid degradation (followed by monitoring of function), alter its activity, and look for loosely-associated protein partners by cross-linking and pull-downs.

Summary

Polyphophoinositol lipids are minor components (about 1%) of the phospholipids that make up mammalian cell membranes, but they have a physiological and pathological importance way out of proportion to their quantity. There are seven of them in all, each with at least one (most with more than one) physiological action to regulate how cells function - and these actions take place in almost every conceivable location within a cell. When these processes are not appropriately regulated, pathologies can occur: polyphosphoinositol lipids are implicated in, for example, cancer, inflammation, neurological disorders and cardiovascular disease. One subject of this application is the most recently discovered polyphosphoinositol lipid, phosphatidylinositol 5-phophate (PI5P), which is present in many locations of the cell and which has, by a sigificant body of circumstantial evidence, been implicated in the way in which cells respond to stress (uv light, heat, oxidising radicals), responses which self-evidently must act as part of their defence against environmentally-induced disease. The levels of PI5P (and thus its functions, because its actions are regulated primarily by how much is present at a given location) are regulated by a family of enzymes: the PI5P 4-kinases (PI5P4Ks). Humans have three PI5P4K genes (and thus three different PI5P4Ks), and one of these, PI5P4K gamma, has been ignored almost completely so far (except by us). Yet we have found that it has a striking expression pattern, being found at very high levels in precise and specific locations in epithelial cells in the kidney, and also in neurons within particular parts of the brain and the spinal cord. Inside the cells that express PI5P4K gamma it appears to be attached to membrane vesicles within the cytoplasm, but what these vesicles are, and what they are doing is unclear (and clarification is one of the major aims of this study). To further improve on our paucity of understanding of PI5P4K gamma we plan toapply many of the cutting edge tools of molecular cell biology to its study. We are in the final stages of generating a mouse lacking the PI5P4K gamma gene, and how this mouse functions in its kidneys and nervous system may give us new insights. Even if they do not, the availability of tissues and embryonic stem cells from these mice will give us powerful new tools to understand exactly where this protein is functioning and what it is doing. Our collaborators in this aspect will also investigate how removal of the PI5P4K gamma in zebra fish embryos alters the form and function of kidneys and neurons in this organism. In parallel we will use cell lines derived from the same part of the kidney that expresses PI5P4K gamma highly and also we will make full use of a cell line called Nalm-6 cells, which is derived from a human B cell lymphoma. The unique properties of Nalm-6 cells (it is extremely easy to manipulate genes in them) will enable us to 'tag', remove, and change PI5P4K gamma in order to find out who it talks to, and where, and what it says.

Impact Summary

(a) The impact of this application is largely scientific, in that it seeks to make important contributions to our basic understanding of how cellular stress pathways operate. Inositol lipids have a wide range of crucial physiological functions, and although PI5P4Ks are generally thought to be involved in cellular stress signalling, this is not certain, and any attempt to try to clarify what they do should be to the benefit of our wider understanding of animal pathologies. (b) There is a potential societal impact in that if the PI5P4K gamma k.o. mice do lead directly to insights into kidney or brain physiology, then there is a small but significant chance that a rapid transfer of information to improving stressed kidney physiological responses or neuronal malfunction could ensue.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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