Membrane-associated phospholipase C of Drosophila retina

Phospholipase C activities against phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol have been examined using head homogenate of Drosophila visual mutants. In many mutants the enzyme activities were found to be reduced. The activities against both phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol were always affected in parallel among the mutants, while the activities of other enzymes related to phosphatidylinositol metabolism, such as diacylglycerol kinase, were not. The enzyme was concluded to be membrane-associated and was activated maximally at low Ca2+ concentration (10(-7) M), when phosphatidylinositol 4,5-bisphosphate was used as a substrate, while the activity obtained with phosphatidylinositol increased with the Ca2+ concentration up to 10(-4) M. The effects of pH on these two enzyme activities differed to some extent.     

Phosphorylation of phosphatidylinositol by transverse tubule vesicles and its possible role in excitation-contraction coupling

Phosphorylation of phosphatidylinositol to phosphatidylinositol 4-monophosphate and to phosphatidylinositol 4,5-bisphosphate was demonstrated in transverse-tubule membranes isolated from frog skeletal muscle using [gamma-32P]ATP as substrate. At millimolar concentrations of Mg2+ both phosphorylation reactions were completed within 15 s at 25 degrees C. Isolated sarcoplasmic reticulum vesicles phosphorylated phosphatidylinositol to phosphatidylinositol 4-phosphate with a lower specific activity than the transverse tubules, and lacked the ability to produce phosphatidylinositol 4,5-bisphosphate. These findings show, for the first time, that isolated transverse-tubule membranes carry out one of the steps required to sustain a role for inositol trisphosphate as the physiological messenger in excitation-contraction coupling in skeletal muscle. The finding that 0.5 mM tetracaine apparently inhibits the phosphorylation of phosphatidylinositol 4-phosphate to phosphatidylinositol 4,5-bisphosphate also supports a role for these intermediates in excitation-contraction coupling.     

Changes in polyphosphoinositide levels in rat liver nuclei in response to prolactin, a known hepatic mitogen.

The effect of prolactin action on nuclear polyphosphoinositide synthesis was investigated in isolated rat liver nuclei. An increased uptake of phosphate from [gamma 32P] adenosinetriphosphate was observed in both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate with a maximum response at 10(-12) M concentration of hormone. Pulse-chase experiments in isolated nuclei following prolactin treatment indicate that the observed increase in accumulation of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate is mainly due to a decrease in their rate of turnover possibly induced by a change in activity of polyphosphoinositide-specific monoesterases. In vitro prolactin also reduces the activity of nuclear phospholipase C specific for phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Moreover, this feature is strongly supported by the concomitant decrease in nuclear diacylglycerol mass. Thus these data suggest that once prolactin reaches the nucleus an intranuclear signalling is evoked through inositol lipid metabolism.     

The polyphosphoinositide phosphodiesterase of erythrocyte membranes

1. A new assay procedure has been devised for measurement of the Ca(2+)-activated polyphosphoinositide phosphodiesterase (phosphatidylinositol polyphosphate phosphodiesterase) activity of erythrocyte ghosts. The ghosts are prepared from cells previously incubated with [(32)P]P(i). They are incubated under appropriate conditions for activation of the phosphodiesterase and the released (32)P-labelled inositol bisphosphate and inositol trisphosphate are separated by anion-exchange chromatography on small columns of Dowex-1 (formate form). When necessary, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate can be deacylated and the released phosphodiesters separated on the same columns. 2. The release of both inositol bisphosphate and inositol trisphosphate was rapid in human ghosts, with half of the labelled membrane-bound phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate broken down in only a few minutes in the presence of 0.5mm-Ca(2+). For both esters, optimum rates of release were seen at pH6.8-6.9. Mg(2+) did not provoke release of either ester. 3. Ca(2+) provoked rapid polyphosphoinositide breakdown in rabbit erythrocyte ghosts and a slower breakdown in rat ghosts. Erythrocyte ghosts from pig or ox showed no release of inositol phosphates when exposed to Ca(2+). 4. In the presence of Mg(2+), the inositol trisphosphate released from phosphatidylinositol 4,5-bisphosphate was rapidly converted into inositol bisphosphate by phosphomonoesterase activity. 5. Neomycin, an aminoglycoside antibiotic that interacts with polyphosphoinositides, inhibited the breakdown of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, with the latter process being appreciably more sensitive to the drug. Phenylmethanesulphonyl fluoride, an inhibitor of serine esterases that is said to inhibit phosphatidylinositol phosphodiesterase, had no effect on the activity of the erythrocyte polyphosphoinositide phosphodiesterase. 6. These observations are consistent with the notion that human, and probably rabbit and rat, erythrocyte membranes possess a single polyphosphoinositide phosphodiesterase that is activated by Ca(2+) and that attacks phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate with equal facility. Inhibition of this activity by neomycin seems likely to be due to interactions between neomycin and the polyphosphoinositides, with the greater inhibition of phosphatidylinositol 4,5-bisphosphate breakdown consistent with the greater affinity of the drug for this lipid. In addition, erythrocyte membranes possess Mg(2+)-dependent phosphomonoesterase that converts inositol 1,4,5-triphosphate into inositol bisphosphate.     

Phosphatidyl inositol 4,5-bisphosphate-specific phospholipase C in bovine rod outer segment membranes.

Polyphosphoinositide-specific phosphodiesterase (phospholipase C) activity against phosphatidylinositol 4,5-bisphosphate has been examined in disrupted bovine retinal rod outer segments. The enzyme was strictly modulated by free calcium ion concentration and maximally activated at 10(-5) M Ca2+ (91 +/- 4 nmoles phosphatidylinositol 4,5-bisphosphate hydrolyzed/min/mg of protein). Guanine nucleotides did not affect in vitro phospholipase C activity either in the presence or absence of light, carbachol or epinephrine. The pH optimum at 10(-5) M Ca2+ in the presence of sodium deoxycholate was 6.5. The enzyme of bovine rod outer segments was concluded to be indirectly regulated by the phototransduction events.     

Rapid and transient thrombin stimulation of phosphatidylinositol 4,5-bisphosphate synthesis but not of phosphatidylinositol 3,4-bisphosphate independent of phospholipase C activation in platelets

When platelets are stimulated by thrombin they immediately undergo inositol lipid hydrolysis via phospholipase C activation. However, subsequently an increased production of phosphatidylinositol 4,5-bisphosphate is observed. Phospholipases C were inhibited by lowering the cytoplasmic free calcium concentration by preincubation with Quin-2-tetra(acetoxymethyl) ester. Aggregation and secretion were also totally suppressed. Under these conditions we observed an increased labeling of phosphatidylinositol 4,5-bisphosphate, indicating a stimulation of inositol lipid kinases, independent of lipid hydrolysis by phospholipase C. Conversely the production of phosphatidylinositol 3,4-bisphosphate was totally abolished. These results suggest a different regulation of the kinases/phosphatases responsible for the production of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate.     

Simulations of Kindlin-2 PIP binding domains reveal protonation-dependent membrane binding modes

Kindlin-2, a member of the Kindlin family of peripheral membrane proteins, is important for integrin activation and stabilization of epidermal growth factor receptor. It associates with the cytoplasmic face of the plasma membrane via dedicated phosphatidylinositol phosphate binding domains located in the N-terminal F0 and Pleckstrin Homology domains. These domains have binding affinity for phosphatidylinositol 4,5-bisphosphate and, to a greater degree, phosphatidylinositol 3,4,5-trisphosphate. The biological significance of the differential binding of these phosphatidylinositol phosphates to Kindlin-2 and the mechanism by which they activate Kindlin-2 are not well understood. Recently, ssNMR identified the predominant protonation states of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate near physiological pH in the presence of anionic lipids. Here, we perform atomistic simulation of the bound state of the Pleckstrin Homology and F0 domains of Kindlin-2 at membranes containing phosphatidylinositol 4,5-bisphosphate/phosphatidylinositol 3,4,5-trisphosphate with differing protonation states. This computational approach demonstrates that these two phosphatidylinositol phosphates differently modulate Kindlin-2 subdomain binding in a protonation-state-dependent manner. We speculate these variations in binding mode provide a mechanism for intracellular pH and Ca²⁺ influx to control the membrane binding behavior and activity of Kindlin-2.     

Phosphatidylinositol 4,5-bisphosphate phosphodiesterase in higher plants.

A phospholipase C which hydrolyses phosphatidylinositol 4,5-bisphosphate to release inositol trisphosphate was detected in a sedimentable fraction from celery and from some other higher plants. The particulate enzyme also hydrolyses phosphatidylinositol, whereas the soluble phosphatidylinositol phosphodiesterase described previously [Irvine, Letcher & Dawson (1980) Biochem. J. 192, 279-283] acts only on phosphatidylinositol, and we were unable to detect activity of this soluble activity on phosphatidylinositol 4,5-bisphosphate. Activity of the particulate enzyme is markedly enhanced in the presence of deoxycholate, but not of other detergents; the particulate enzyme can also be solubilized by extraction with deoxycholate.     

Regulation of plasma membrane Ca2+ ATPases by lipids of the phosphatidylinositol cycle

When the erythrocyte plasma membrane Ca2+ pump is reconstituted into phosphatidylcholine liposomes, the inclusion of small amounts of phosphatidic acid or phosphatidylinositol 4,5-bisphosphate stimulates the enzyme's activity. Other lipids of the phosphatidylinositol cycle (diacylglycerol, phosphatidylinositol) have little effect. The stimulatory effect of phosphatidylinositol 4,5-bisphosphate is greater than that of calmodulin; this lipid also stimulates the plasma membrane Ca2+ ATPase from rat brain.     

Subcellular localisation of inositol lipid kinases in rat liver

The subcellular distribution of the enzymes which phosphorylate phosphatidylinositol sequentially to form phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was investigated in rat liver. We demonstrate that whilst phosphatidylinositol kinase is present in Golgi, lysosomes and plasma membranes, the kinase that forms phosphatidylinositol 4,5-bisphosphate is localised predominantly at the plasma membrane. The role of the inositol lipid kinases in cell function is discussed.     

Identification and characterization of a sac domain-containing phosphoinositide 5-phosphatase

We have characterized a novel Sac domain-containing inositol phosphatase, hSac2. It was ubiquitously expressed but especially abundant in the brain, heart, skeletal muscle, and kidney. Unlike other Sac domain-containing proteins, hSac2 protein exhibited 5-phosphatase activity specific for phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. This is the first time that the Sac domain has been reported to possess 5-phosphatase activity. Its 5-phosphatase activity for phosphatidylinositol 4,5-bisphosphate (K(m) = 14.3 microm) was comparable with those of Type II 5-phosphatases. These results imply that hSac2 functions as an inositol polyphosphate 5-phosphatase.     

Nuclear translocation of phospholipase C-delta1 is linked to the cell cycle and nuclear phosphatidylinositol 4,5-bisphosphate

Nuclear phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate, fluctuate throughout the cell cycle and are linked to proliferation and differentiation. Here we report that phospholipase C-delta(1) accumulates in the nucleus at the G(1)/S boundary and in G(0) phases of the cell cycle. Furthermore, as wild-type protein accumulated in the nucleus, nuclear phosphatidylinositol 4,5-bisphosphate levels were elevated 3-5-fold, whereas total levels were decreased compared with asynchronous cultures. To test whether phosphatidylinositol 4,5-bisphosphate binding is important during this process, we introduced a R40D point mutation within the pleckstrin homology domain of phospholipase C-delta(1), which disables high affinity phosphatidylinositol 4,5-bisphosphate binding, and found that nuclear translocation was significantly reduced at G(1)/S and in G(0). These results demonstrate a cell cycle-dependent compartmentalization of phospholipase C-delta(1) and support the idea that relative levels of phosphoinositides modulate the portioning of phosphoinositide-binding proteins between the nucleus and other compartments.     

Regulation of ion channels by phosphatidylinositol 4,5-bisphosphate

Phosphatidylinositol 4,5-bisphosphate is a signaling phospholipid of the plasma membrane that has a dynamically changing concentration. In addition to being the precursor of inositol trisphosphate and diacylglycerol, it complexes with and regulates many cytoplasmic and membrane proteins. Recent work has characterized the regulation of a wide range of ion channels by phosphatidylinositol 4,5-bisphosphate, helping to redefine the role of this lipid in cells and in neurobiology. In most cases, phosphatidylinositol 4,5-bisphosphate increases channel activity, and its hydrolysis by phospholipase C reduces channel activity.     

Gelsolin in complex with phosphatidylinositol 4,5-bisphosphate inhibits caspase-3 and -9 to retard apoptotic progression

Apoptosis, or programmed cell death, occurs because of the activation of a protease cascade amplification circuit that includes the critical effector caspase-3. Previously, we identified the widely expressed actin modulatory protein gelsolin as a prominent substrate of caspase-3 and demonstrated that the N-terminal gelsolin cleavage product promotes apoptosis. Here we show that phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3, 4-bisphosphate in pure micelles or mixed vesicles prevent caspase-3 cleavage of gelsolin. Moreover, phosphatidylinositol 4, 5-bisphosphate-gelsolin strongly inhibits caspase-3 and -9 activity through the formation of a stable phosphatidylinositol 4, 5-bisphosphate-gelsolin-caspase complex. In addition, phosphatidylinositol 4,5-bisphosphate-gelsolin prevents apoptotic progression mediated by caspase-3 in a cell-free system, and phosphatidylinositol 4,5-bisphosphate-gelsolin-caspase-9 and phosphatidylinositol 4,5-bisphosphate-gelsolin-caspase-3 complexes form in mouse embryonic fibroblasts during apoptosis induction when stimulated with fibronectin, to delay cell death. The results suggest that gelsolin can act as both an effector and an inhibitor of caspase-3, the latter in concert with phosphatidylinositol 4, 5-bisphosphate, and other membrane phospholipids to regulate the onset and progression of apoptosis.     

A central kinase domain of type I phosphatidylinositol phosphate kinases is sufficient to prime exocytosis: isoform specificity and its underlying mechanism

Exocytosis, a critical process for neuronal communication and hormonal regulation, involves several distinct steps including MgATP-dependent priming (which involves the synthesis of phosphatidylinositol 4,5-bisphosphate). Type I phosphatidylinositol phosphate kinases (PIPKIs) were purified biochemically as a priming factor. PIPKI consists of three domains: the N-terminal region, the central kinase domain, and the C-terminal region. Three isoforms (alpha, beta, and gamma) of PIPKI have been identified, and each is alternatively spliced at the C-terminal region. In the present study, we conducted a structure/function analysis of PIPKIs in the priming of exocytosis, and we found that recombinant PIPKIalpha and PIPKIgamma had priming activity. However, an unexpected finding of these results was that PIPKIbeta did not prime exocytosis. The N- or C-terminal region of PIPKIalpha and PIPKIgamma was not required for priming, which indicates that the central kinase domain is sufficient for this process. Alternative splicing in each isoform did not affect the isoform specificity in priming. Priming activity by isoforms is strongly correlated with their phosphatidylinositol phosphate kinase activity because PIPKIalpha and PIPKIgamma had higher kinase activity than PIPKIbeta. These results suggest that PIPKIalpha and PIPKIgamma are the critical priming factors for exocytosis; it also suggests that the levels of phosphatidylinositol phosphate kinase activity in producing phosphatidylinositol 4,5-bisphosphate specify the function of PIPKI isoforms in priming.     

Purification and characterization of a phosphoinositide-specific phospholipase C from guinea pig uterus. Phosphorylation by protein kinase C in vivo

Two peaks of phosphoinositide-specific phospholipase C (PI-PLC) activity were resolved when guinea pig uterus cytosolic proteins were chromatographed on a DEAE-Sepharose column. The first peak of enzyme activity eluting from the DEAE-Sepharose column (PI-PLC I) was further purified to homogeneity, whereas the second peak of enzyme activity was enriched 300-fold. PI-PLC I migrated as a 62-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. Antibodies prepared against PI-PLC I failed to react with PI-PLC II. PI-PLC I hydrolyzed all three phosphoinositides, exhibiting a greater Vmax for phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. Hydrolysis of phosphatidylinositol was calcium-dependent, whereas significant hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate occurred in the presence of 2.5 mM EGTA. At physiological concentrations of calcium, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were the preferred substrates. Antibodies specific for PI-PLC I reacted with a 62-kDa protein in both the cytosol and membrane fractions from guinea pig uterus. Quantitation of the immunoblots revealed that 25% of the 62-kDa protein was membrane-associated, whereas only 5% of the total enzyme activity was membrane-associated. Approximately 20% of the membrane-bound phospholipase C activity and immunoreactive material were loosely bound, whereas the remainder required detergent extraction for complete solubilization. The 62-kDa protein associated with the membrane fractions did not bind lectin affinity columns, suggesting that it was not glycosylated. PI-PLC I was identified as a phosphoprotein in [32P]orthophosphate-labeled rat basophilic leukemia (RBL-1) cells by two-dimensional gel electrophoresis followed by immunoblotting. In untreated cells, 32P-labeled PI-PLC I was found in the cytosolic fraction. Treatment of RBL-1 cells with those phorbol esters which are known to activate the Ca2+/phospholipid-dependent enzyme protein kinase C, resulted in a time-dependent increase in the phosphorylation of both membrane-bound and cytosolic PI-PLC I. Thus, in RBL-1 cells, protein kinase C may play an important role in the regulation of phospholipase C through protein phosphorylation.     

Phosphoinositide synthesis and Ca2+ gating in blowfly salivary glands exposed to 5-hydroxytryptamine.

Blowfly salivary glands, previously exposed to 10 microM-5-hydroxytryptamine for 30 min, demonstrated a rapid compensatory resynthesis of [3H]inositol-labelled phosphatidylinositol 4,5-bisphosphate when allowed to recover in medium containing 3-5 microM-inositol. Phosphatidylinositol 4,5-bisphosphate comprised 70% of the total [3H]-phosphoinositide, and there was a corresponding decrease in the formation of [3H]-phosphatidylinositol. Subsequent addition of 5-hydroxytryptamine produced an equivalent breakdown of the newly synthesized phosphoinositides but little 45Ca2+ gating. Increasing the inositol concentration in the medium to 300 microM produced a 14-fold stimulation of phosphatidylinositol synthesis but only a 5-fold increase in phosphatidylinositol 4,5-bisphosphate synthesis. Increasing the inositol concentration in the medium from 3 microM to 300 microM resulted in a progressively greater recovery of the 45Ca2+-gating response. At 300 microM-inositol there was an 85% recovery of 45Ca2+-gating response. These results indicate that conversion of phosphatidylinositol into phosphatidylinositol 4,5-bisphosphate occurs in blowfly salivary glands and is secondary to an initial breakdown of the phosphoinositides. Recovery of Ca2+ gating is dependent on the restoration of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate to appropriate concentrations.     

Characterization of partially purified phospholipase C from human platelet membranes.

A phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-hydrolytic activity was found to be present in the human platelet membrane fraction, with 20% of the total activity of the homogenate. The membrane-associated phospholipase C activity was extracted with 1% deoxycholate (DOC). The DOC-extractable phospholipase C was partially purified approx. 126-fold to a specific activity of 0.58 mumol of PtdIns-(4,5)P2 cleaved/min per mg of protein, by Q-Sepharose, heparin-Sepharose and Ultrogel AcA-44 column chromatographies. This purified DOC-extractable phospholipase C had an Mr of approx. 110,000, as determined by Ultrogel AcA-44 gel filtration. The enzyme exhibits a maximal hydrolysis for PtdIns-(4,5)P2 at pH 6.5 in the presence of 0.1% DOC. The addition of 0.1% DOC caused a marked activation of both PtdIns(4,5)P2 and phosphatidylinositol (PtdIns) hydrolyses by the enzyme. The enzyme hydrolysed PtdIns(4,5)P2 and PtdIns in a different Ca2+-dependent manner; the maximal hydrolyses for PtdIns(4,5)P2 and PtdIns were obtained at 4 microM- and 0.5 mM-Ca2+ respectively. In the presence of 1 mM-Mg2+, PtdIns(4,5)P2-hydrolytic activity was decreased at all Ca2+ concentrations examined, but PtdIns-hydrolytic activity was not affected.     

Isolation and characterization of rat 3Y1 fibroblast clones overexpressing the src homology region of phospholipase C-gamma 2.

To examine the regulatory function of the src-related SH2 and SH3 (SH2/SH3) region of phospholipase C-gamma 2 (PLC-gamma 2), we expressed this region of rat PLC-gamma 2 cDNA in rat 3Y1 fibroblasts and isolated and characterized a number of clones (approximately 20 clones). An increase of endogenous tyrosine kinase activity was observed in all cell clones that highly expressed a translational product of the SH2/SH3 domain. Moreover, endogenous phosphatidylinositol 4,5-bisphosphate hydrolyzing activity was also enhanced in these clones, and PLC-gamma 1 seemed to be preferentially activated among endogenous PLC isozymes. Genistein, an inhibitor of tyrosine kinase, inhibited this activation of PLC-gamma 1, and tyrosine phosphorylation was observed on PLC-gamma 1 molecules, indicating the involvement of tyrosine kinases in the PLC-gamma 1 activation. These results suggest that the SH2/SH3 region of PLC-gamma would function as a multidirectional regulator which controls at least two major signaling pathways: tyrosine kinase and phosphatidylinositol 4,5-bisphosphate hydrolysis.     

Autophosphorylation of type I phosphatidylinositol phosphate kinase regulates its lipid kinase activity

Phosphatidylinositol phosphate kinases (PIPKs) have important roles in the production of various phosphoinositides. For type I PIP5Ks (PIP5KI), a broad substrate specificity is known. They phosphorylate phosphatidylinositol 4-phosphate most effectively but also phosphorylate phosphatidylinositol (PI), phosphatidylinositol 3-phosphate, and phosphatidylinositol (3,4)-bisphosphate (PI(3, 4)P(2)), resulting in the production of phosphatidylinositol (4, 5)-bisphosphate (PI(4,5)P(2)), phosphatidylinositol 3-phosphate, phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P(2)), phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P(2)), and phosphatidylinositol (3,4,5)-trisphosphate. We show here that PIP5KIs have also protein kinase activities. When each isozyme of PIP5KI (PIP5KIalpha, -beta, and -gamma) was subjected to in vitro kinase assay, autophosphorylation occurred. The lipid kinase-negative mutant of PIP5KIalpha (K138A) lost the protein kinase activity, suggesting the same catalytic mechanism for the lipid and the protein kinase activities. PIP5KIbeta expressed in Escherichia coli also retains this protein kinase activity, thus confirming that no co-immunoprecipitated protein kinase is involved. In addition, the autophosphorylation of PIP5KI is markedly enhanced by the addition of PI. No other phosphoinositides such as phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, or phosphatidylinositol trisphosphate have such an effect. We also found that the PI-dependent autophosphorylation strongly suppresses the lipid kinase activity of PIP5KI. The lipid kinase activity of PIP5KI was decreased to one-tenth upon PI-dependent autophosphorylation. All these results indicate that the lipid kinase activity of PIP5KI that acts predominantly for PI(4,5)P(2) synthesis is regulated by PI-dependent autophosphorylation in vivo.