Type I PIPkins (phosphatidylinositol 4-phosphate 5-kinases) are the enzymes that catalyse the major cellular route of synthesis of PtdIns(4,5) P2, and three isoforms (alpha, beta and gamma) with several splice variants have been found to date. In the present paper, we describe the discovery of a novel splice variant of the gamma isoform, which we call PIPkin Igammac, and which is characterized by the inclusion of a 26-amino-acid insert near the C-terminus. Its transcript appears to be selectively expressed in brain, where it locates in the neurons of restricted regions, such as cerebellum, hippocampus, cortex and olfactory bulb, as indicated by in situ hybridization studies. Overexpression of two different catalytically inactive constructs of PIPkin Igammac in rat cerebellar granule cells causes a progressive loss of their neuronal processes, whereas equivalent kinase-dead versions of PIPkin Igammaa did not induce any such effect, suggesting the possible existence of a specific PtdIns(4,5) P2 pool synthesized by PIPkin Igammac, which is involved in the maintenance of some neuronal cellular processes.

The family of phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) is emerging from a comparative backwater in inositide signalling into the mainstream, as is their substrate, phosphatidylinositol 5-phosphate (PI5P). Here we review some of the key questions about the PI5P4Ks, their localisation, interaction, and regulation and also we summarise our current understanding of how PI5P is synthesised and what its cellular functions might be. Finally, some of the evidence for the involvement of PI5P4Ks in pathology is discussed.

Cellular adhesion can be regulated by, as yet, poorly defined intracellular signalling events. Phospholipase D enzymes generate the messenger lipid phosphatidate and here we demonstrate that suppression of this reaction inhibits cellular adhesion. This effect was reversed by the addition of cell-permeable analogues of either phosphatidate or phosphatidylinositol 4,5-bisphosphate. By contrast, neither diacylglycerol nor lysophosphatidic acid were able to reverse this effect suggesting that phosphatidate itself acts directly on a target protein(s) to regulate adhesion rather than as the result of its conversion to either of these metabolite lipids. Antibodies that block β1 and β2 integrin-substrate interactions inhibited adhesion stimulated by both phosphatidate and phosphatidylinositol 4,5-bisphosphate indicating that these lipids regulate β1 and β2 integrin-mediated adhesion. In vivo, these lipids can be generated by phospholipase D2 and phosphatidylinositol 4-phosphate 5-kinase Iγb, respectively, and over-expression of catalytically-functional forms of these enzymes dose-dependently stimulated adhesion while siRNA depletion of PLD2 levels inhibited adhesion. Furthermore the ability of over-expressed phospholipase D2 to stimulate adhesion was inhibited by a dominant-negative version of phosphatidylinositol 4-phosphate 5-kinase Iγb. Consistent with this, phosphatidylinositol 4-phosphate 5-kinase Iγb-mediated adhesion was dependent upon phospholipase D2's product, phosphatidate indicating that phosphatidylinositol 4-phosphate 5-kinase Iγb is downstream of, and necessary for, phospholipase D2's regulation of adhesion. It is likely that this phospholipase D2-generated phosphatidate directly stimulates phosphatidylinositol 4-phosphate 5-kinase Iγb to generate phosphatidylinositol 4,5-bisphosphate as this mechanism has previously been demonstrated in vitro. Thus, our data indicates that during the initial stages of adhesion, phospholipase D2-derived phosphatidate stimulates phosphatidylinositol 4-phosphate 5-kinase Iγb to generate phosphatidylinositol 4,5-bisphosphate and that consequently this inositol phospholipid promotes adhesion through its regulation of cell-surface integrins.

NIH-12848 (NCGC00012848-02), a putative phosphatidylinositol 5-phosphate 4-kinase γ (PI5P4Kγ) inhibitor, was explored as a tool for investigating this enigmatic, low activity, lipid kinase. PI5P4K assays in vitro showed that NIH-12848 inhibited PI5P4Kγ with an IC50 of approximately 1 μM but did not inhibit the α and β PI5P4K isoforms at concentrations up to 100 μM. A lack of inhibition of PI5P4Kγ ATPase activity suggested that NIH-12848 does not interact with the enzyme's ATP-binding site and direct exploration of binding using hydrogen–deuterium exchange (HDX)-MS (HDX-MS) revealed the putative PI5P-binding site of PI5P4Kγ to be the likely region of interaction. This was confirmed by a series of mutation experiments which led to the identification of a single PI5P4Kγ amino acid residue that can be mutated to its PI5P4Ks α and β homologue to render PI5P4Kγ resistant NIH-12848 inhibition. NIH-12848 (10 μM) was applied to cultured mouse principal kidney cortical collecting duct (mpkCCD) cells which, we show, express PI5P4Kγ that increases when the cells grow to confluence and polarize. NIH-12848 inhibited the translocation of Na+/K+-ATPase to the plasma membrane that occurs when mpkCCD cells grow to confluence and also prevented reversibly their forming of ‘domes’ on the culture dish. Both these NIH-12848-induced effects were mimicked by specific RNAi knockdown of PI5P4Kγ, but not that of PI5P4Ks α or β. Overall, the data reveal a probable contribution of PI5P4Kγ to the development and maintenance of epithelial cell functional polarity and show that NIH-12848 is a potentially powerful tool for exploring the cell physiology of PI5P4Ks.