Abstract
Phosphatidylinositol 5 phosphate 4-kinase (PIP4K) are enzymes that catalyse the phosphorylation of phosphatidylinositol 5-phosphate (PI5P) to generate PI(4,5)P2. Mammalian genomes contain three genes, PIP4K2Α, 2B and 2C and murine knockouts for these suggested important physiological roles in vivo. The proteins encoded by PIP4K2A, 2B and 2C show widely varying specific activities in vitro; PIP4K2A is highly active and PIP4K2C 2000-times less active, and the relationship between this biochemical activity and in vivo function is unknown. By contrast, the Drosophila genome encodes a single PIP4K (dPIP4K) that shows high specific activity in vitro and loss of this enzyme results in reduced salivary gland cell size in vivo. We find that the kinase activity of dPIP4K is essential for normal salivary gland cell size in vivo. Despite their highly divergent specific activity, we find that all three mammalian PIP4K isoforms are able to enhance salivary gland cell size in the Drosophila PIP4K null mutant implying a lack of correlation between in vitro activity measurements and in vivo function. Further, the kinase activity of PIP4K2C, reported to be almost inactive in vitro, is required for in vivo function. Our findings suggest the existence of unidentified factors that regulate PIP4K enzyme activity in vivo.
Highlights
The phosphorylation of lipids to generate phospho-variants is a strategy used to generate signalling molecules that encode information on ongoing cellular processes
phosphatidylinositol 4-phosphate (PI4P) and PI(4,5)P2 are substantially more abundant in eukaryotic cells and previous studies have suggested that the bulk of the PI(4,5)P2 in animal cells is synthesised by this pathway [3] and (ii) the phosphorylation of phosphatidylinositol 5-phosphate (PI5P) at position 4 of the inositol head group; the enzymes that catalyse this reaction are the phosphatidylinositol 5 phosphate 4-kinases (PIP4K)
While the C. elegans enzyme appears to have very low specific activity in vitro, recapitulating that seen for hPIP4K2C, the Drosophila enzyme appears to have high specific activity similar to hPIP4K2A
Summary
The phosphorylation of lipids to generate phospho-variants is a strategy used to generate signalling molecules that encode information on ongoing cellular processes. While the single PIP4K in the Drosophila genome shows high specific activity comparable with PIP4K2A, the only PIP4K gene in C. elegans (ppk-2) shows very low activity similar to that seen for PIP4K2C [14] The reason for this widely varying activity of PIP4K as measured in vitro and its relevance to in vivo functions remains unclear. We reconstituted PIP4K function in the salivary glands of dPIP4K29, a loss of function mutant in the single dPIP4K Such reconstitution with dPIP4K was able to rescue the cell size phenotype and this rescue was dependent on the kinase activity of the enzyme. Further we find that all isoforms of mammalian PIP4K, irrespective of their specific activity as measured in vitro, were individually able to rescue the reduced cell size phenotype of dPIP4K29. Additional factors in intact cells may regulate the activity of PIP4K enzymes
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