Inhibitors of PI(4,5)P₂Synthesis Reveal Dynamic Regulation of IgE Receptor Signaling by Phosphoinositides in RBL Mast Cells

Abstract

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) is a versatile phospholipid that participates in many membrane-associated signaling processes. PI(4,5)P 2 production at the plasma membrane (PM) depends on levels of its precursor, phosphatidylinositol 4-phosphate (PI4P), synthesized principally by two intracellular enzymes, PI4-kinases IIIa and IIIb; the former is preferentially inhibited by phenylarsine oxide (PAO). We found that PAO and quercetin, another lipid kinase inhibitor, rapidly inhibit Ca 21 responses to antigen in IgE-sensitized rat basophilic leukemia mast cells. Quercetin also rapidly inhibits store-operated Ca 21 influx stimulated by thapsigargin. In addition, quercetin and PAO effectively inhibit antigen-stimulated ruffling and spreading in these cells, and they inhibit endocytosis of crosslinked IgE receptor complexes, evidently by inhibiting pinching off of endocytic vesicles containing the clustered IgE receptors. A minimal model to account for these diverse effects is inhibition of PI(4,5)P 2 synthesis by PAO and quercetin. To characterize the direct effects of these agents on PI(4,5)P 2 synthesis, we monitored the reappearance of the PI(4,5)P 2 -specific PH domain PH-phospholipase C d-EGFP at the PM after Ca 21 ionophore (A23187)-induced PI(4,5)P 2 hydrolysis, followed by Ca 21 chelation with excess EGTA. Resynthesized PI(4,5)P 2 initially appears as micron-sized patches near the PM. Addition of quercetin subsequent to A23187-induced PI(4,5)P 2 hydrolysis reduces PI(4,5)P 2 resynthesis in PM-associated patches, and PAO reduces PI(4,5)P 2 at the PM while enhancing PI(4,5)P 2 accumulation at the Golgi complex. Taken together, these results provide evidence that PI4P generated by PI4-kinase IIIa is dynamically coupled to PI(4,5)P

Nanodomains in early and later phases of FcɛRI signalling

“…We observed parallel inhibition of the stimulated association between oligomeric STIM1 and Orai1, as monitored by fluorescence resonance energy transfer (FRET) between fluorescent protein-tagged constructs [21, 22]. This stimulated FRET was also decreased by 10 μM wortmannin, which inhibits several different phosphoinositide kinases important in Ca 2+ signaling [23, 24]. …”

“…In a recent study, we gained distributional information while investigating the effectiveness of inhibitors of PI-kinases in interfering with signaling responses downstream of FcεRI activation in RBL cells. In particular, we observed that re-synthesis of PIP 2 at the plasma membrane following its hydrolysis (stimulated by high concentrations of cytoplasmic Ca 2+ ) occurred in discrete puncta at or near the plasma membrane, as monitored by appearance of PLCδ1 PH-EGF at these sites on the timescale of several minutes at 37°C [24]. These PIP 2 puncta were often as large as several microns in diameter, were stably localized over this time period.The puncta were co-labeled by fluorescent cholera toxin B, often used as a marker for detergent-resistant, ordered membrane domains, similar to previous observations [65].…”

“…At longer times, the distribution of PLCδ1 PH-EGF became more uniform at the plasma membrane in most cells. Interestingly, both PI-kinase inhibitors, phenyl arsine oxide and quercetin, inhibited the reappearance of PLCδ1 PH-GFP binding to these puncta, suggesting that new synthesis of PIP 2 occurs in these membrane domains [24]. It is not yet clear whether these puncta represent physiologically relevant domains of new PIP 2 synthesis, or whether they result from non-physiological elevation of cytoplasmic Ca 2+ by the Ca 2+ ionophore, A23187, which induces large-scale hydrolysis and re-synthesis of PIP 2 in these cells.…”

A novel fluorescence-based biosynthetic trafficking method provides pharmacologic evidence that PI4-kinase IIIα is important for protein trafficking from the endoplasmic reticulum to the plasma membrane

“…To distinguish whether retention of EGFR in the ER upon PAO treatment is due to phosphatase or PI 4-kinase inhibition, we tested the effects of the PI 4-kinase inhibitors wortmannin and quercetin [2-(3,4-dihydroxy-phenyl)-3,5,7-trihydroxy-4 H -chromen-4-one], neither of which inhibits tyrosine phosphatases at the concentrations used [34,39,40]. …”

Structure-Function Relationships of ErbB RTKs in the Plasma Membrane of Living Cells

“…In addition, these investigators used 20 mm phenyl arsine oxide (PAO) to inhibit endocytosis of the EGFR. PAO is a potent tyrosine phosphatase inhibitor (Garcia-Morales et al 1990), and also specifically inhibits protein kinase IIa, which is responsible for maintaining the balance of PIP2 in the plasma membrane (Santos et al 2013). PIP2 has been shown to influence the activity of the ErbB1 by binding to the intracellular juxtamembrane domain (Michailidis et al 2011).…”