Exploring phosphatidylinositol 5-phosphate 4-kinase function.

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.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.jbior.2014.09.00

Nuclear localizations of phosphatidylinositol 5-phosphate 4-kinases α and β are dynamic and independently regulated during starvation-induced stress.

The chicken B-cell line DT40 has two isoforms of phosphatidylinositol 5-phosphate 4-kinase (PI5P4K), α and β, which are likely to exist as a mixture of obligate homo- and hetero-dimers. Previous work has led us to speculate that an important role of the β isoform may be to target the more active PI5P4Kα isoform to the nucleus. In the present study we expand upon that work by genomically tagging the PI5P4Ks with fluorochromes in the presence or absence of stable or acute depletions of PI5P4Kβ. Consistent with our original hypothesis we find that PI5P4Kα is predominantly (possible entirely) cytoplasmic when PI5P4Kβ is stably deleted from cells. In contrast, when PI5P4Kβ is inducibly removed within 1 h PI5P4Kα retains its wild-type distribution of approximately 50:50 between cytoplasm and nucleus even through a number of cell divisions. This leads us to speculate that PI5P4Kα is chromatin-associated. We also find that when cells are in the exponential phase of growth PI5P4Kβ is primarily cytoplasmic but translocates to the nucleus upon growth into the stationary phase or upon serum starvation. Once again this is not accompanied by a change in PI5P4Kα localization and we show, using an in vitro model, that this is possible because the dimerization between the two isoforms is dynamic. Given this shift in PI5P4Kβ upon nutrient deprivation we explore the phenotype of PI5P4K B-null cells exposed to this stress and find that they can sustain a greater degree of nutrient deprivation than their wild-type counterparts possibly as a result of up-regulation of autophagy.A.D. was supported by Sidney Sussex College, the Cambridge Overseas Trusts and the Säid Foundation, S.J.B. by an A.J. Clarke Studentship of the British Pharmacological Society and J.H.C. by the MRC (Grant RG64071). We thank Ashok Venkitaraman and Gerard Evan for the kind gifts of reagents.This is the author accepted manuscript. The final version is available from Portland Press via http://dx.doi.org/10.1042/BCJ2016038

In B cells, phosphatidylinositol 5-phosphate 4-kinase-α synthesizes PI(4,5)P2 to impact mTORC2 and Akt signaling.

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are enigmatic lipid kinases with physiological functions that are incompletely understood, not the least because genetic deletion and cell transfection have led to contradictory data. Here, we used the genetic tractability of DT40 cells to create cell lines in which endogenous PI5P4Kα was removed, either stably by genetic deletion or transiently (within 1 h) by tagging the endogenous protein genomically with the auxin degron. In both cases, removal impacted Akt phosphorylation, and by leaving one PI5P4Kα allele present but mutating it to be kinase-dead or have PI4P 5-kinase activity, we show that all of the effects on Akt phosphorylation were dependent on the ability of PI5P4Kα to synthesize phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] rather than to remove PI5P. Although stable removal of PI5P4Kα resulted in a pronounced decrease in Akt phosphorylation at Thr308 and Ser473, in part because of reduced plasma membrane PIP3, its acute removal led to an increase in Akt phosphorylation only at Ser473. This process invokes activation primarily of mammalian target of rapamycin complex 2 (mTORC2), which was confirmed by increased phosphorylation of other mTORC2 substrates. These findings establish PI5P4Kα as a kinase that synthesizes a physiologically relevant pool of PI(4,5)P2 and as a regulator of mTORC2, and show a phenomenon similar to the "butterfly effect" described for phosphatidylinositol 3-kinase Iα [Hart JR, et al. (2015) Proc Natl Acad Sci USA 112(4):1131-1136], whereby through apparently the same underlying mechanism, the removal of a protein's activity from a cell can have widely divergent effects depending on the time course of that removal.S.J.B. was supported by an A.J. Clark Studentship from the British Pharmacological Society, A.D. by Sidney Sussex College, the Cambridge Overseas Trust and the Säid Foundation, and J.H.C by the MRC (Grant RG64071).This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Proceedings of the National Academy of Sciences (PNAS)

In B cells Phosphatidylinositol 5-phosphate 4-kinase α\alpha synthesizes PI(4,5)P2_2 to impact on mTORC2 and Akt signalling

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are enigmatic lipid kinases whose physiological functions are incompletely understood, not least because genetic deletion and cell transfection have led to contradictory data. Here we used the genetic tractability of DT40 cells to create cell lines in which endogenous PI5P4K$\alpha$ was removed either stably by genetic deletion, or transiently (within one hour) by tagging the endogenous protein genomically with the auxin degron. In both cases removal impacted upon Akt phosphorylation, and by leaving one PI5P4K$\alpha$ allele present but mutating it to be kinase-dead or to have PI4P 5-kinase activity we show that all the effects on Akt phosphorylation were dependent upon the ability of PI5P4K$\alpha$ to synthesise PI(4,5)P$_2$ rather than to remove PI5P. Whilst stable removal of PI5P4K$\alpha$ resulted in a pronounced decrease in Akt phosphorylation at Thr308 and Ser473, due in part to reduced plasma membrane PIP$_3$, its acute removal led to an increase in Akt phosphorylation only at Ser473. This invokes activation primarily of mTORC2, which was confirmed by increased phosphorylation of other mTORC2 substrates. These findings establish PI5P4K$\alpha$ as a kinase that synthesizes a physiologically relevant pool of PI(4,5)P$_2$ and as a novel regulator of mTORC2. They also show a phenomenon similar to the ‘butterfly effect’ described for PI 3-kinase I$\alpha$ (Hart JR et al PNAS 112 1131-1136; 2015) whereby via apparently the same underlying mechanism the removal of a protein’s activity from a cell can have widely divergent effects, depending upon the time course of that removal.S.J.B. was supported by an A.J. Clark Studentship from the British Pharmacological Society, A.D. by Sidney Sussex College, the Cambridge Overseas Trust and the Säid Foundation, and J.H.C by the MRC (Grant RG64071).This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Proceedings of the National Academy of Sciences (PNAS)

The inositol 5-phosphatase INPP5B regulates B cell receptor clustering and signaling

Upon antigen binding, the B cell receptor (BCR) undergoes clustering to form a signalosome that propagates downstream signaling required for normal B cell development and physiology. BCR clustering is dependent on remodeling of the cortical actin network, but the mechanisms that regulate actin remodeling in this context remain poorly defined. In this study, we identify the inositol 5-phosphatase INPP5B as a key regulator of actin remodeling, BCR clustering, and downstream signaling in antigen-stimulated B cells. INPP5B acts via dephosphorylation of the inositol lipid PI(4,5)P(2) that in turn is necessary for actin disassembly, BCR mobilization, and cell spreading on immobilized surface antigen. These effects can be explained by increased actin severing by cofilin and loss of actin linking to the plasma membrane by ezrin, both of which are sensitive to INPP5B-dependent PI(4,5)P(2) hydrolysis. INPP5B is therefore a new player in BCR signaling and may represent an attractive target for treatment of B cell malignancies caused by aberrant BCR signaling