Phosphorylation Of Phosphatidylinositol Research Articles

Association of the Abl tyrosine kinase with the Trk nerve growth factor receptor

Nerve growth factor (NGF) initiates the majority of its biological effects by promoting the dimerization and activation of the tyrosine kinase receptor TrkA. In addition to rapid increases in the phosphorylation of phosphatidylinositol 3'-kinase (PI 3-kinase) and phospholipase C-gamma and increased ras activity, phosphorylation of c-Crk and paxillin proteins has been observed upon TrkA activation. The c-Abl tyrosine kinase is involved in the control of the axonal cytoskeleton and is known to interact with c-Crk proteins. Here we have tested the possibility that TrkA receptors might form an association with the c-Abl protein. After transfection in 293T cells, TrkA and c-Abl kinases could be coimmunoprecipitated. This interaction did not require TrkA receptors to be autophosphorylated. Mapping analysis indicated that the region of c-Abl association was confined to the juxtamembrane region of TrkA. The interaction of c-Abl with TrkA was also observed in differentiated pheochromocytoma PC12 cells. These results suggest that c-Abl may be recruited to the NGF receptor complex and be involved in regulating specific phosphorylation events that occur during neuronal differentiation.

Adhesion between cells and extracellular matrix with special reference to hepatic stellate cell adhesion to three-dimensional collagen fibers.

Hepatic stellate cells are located in the perisinusoidal space (space of Disse), and extend their dendritic, thin membranous processes and fine fibrillar processes into this space. The stellate cells coexist with a three-dimensional extracellular matrix (ECM) in the perisinusoidal space. In turn the three-dimensional structure of the ECM regulates the proliferation, morphology, and functions of the stellate cell. In this review, the morphology of sites of adhesion between hepatic stellate cells and extracellular matrix is described. Hepatic stellate cells cultured in polystyrene dishes spread well, whereas the cells cultured on or in type I collagen gel become slender and elongate their long cellular processes which adhere directly to the collagen fibers. Cells in type I collagen gel form a large number of adhesive structures, each adhesive area forming a face but not a point. Adhesion molecules, integrins, for the ECM are localized on the cell surface. Elongation of the cellular processes occurs via integrin-binding to type I collagen fibers. The signal transduction mechanism, including protein and phosphatidylinositol phosphorylation, is critical to induce and sustain the cellular processes. Information on the three-dimensional structures of ECM is transmitted via three-dimensional adhesive structures containing the integrins.

Fluoride-induced depletion of polyphosphoinositides in rat brain cortical slices: a rationale for the inhibitory effects on phospholipase C

Fluoride, which is used commonly as a pharmacological tool to activate phosphoinositide-phospholipase C coupled to the heterotrymeric Gq/11 proteins, inhibited the phosphorylation of phosphatidylinositol (PtdIns) to polyphosphoinositides (PtdIns4P and PtdIns4,5P2) in membranes from rat brain cortex. Fluoride enhanced basal production of 3H-inositol phosphates in membranes prepared from brain cortical slices that had been prelabeled with [3H]inositol, but inhibited the stimulation elicited by carbachol in the presence of GTPγS. However in both cases fluoride depleted [3H]PtdIns4P content by 95%. The inhibitory effects of fluoride on the release of 3H-inositol phosphates in slices were not apparent in a pulse [3H]inositol-labeling strategy, but became dramatic in a continuous labeling protocol, particularly at long incubation times. Prelabeling slices with [3H]inositol in the presence of fluoride precluded polyphosphoinositide labeling, and eliminated phospholipase C responsiveness to carbachol under normal or depolarizing conditions, and to the calcium ionophore ionomycin. The lack of response of 3H-polyphosphoinositide-depleted slices to phospholipase C stimuli was not due to fluoride poisoning, unaccesibility of the [3H]inositol label to phospholipase C or desensitization of Gq/11, as the effect of carbachol and GTPγS was restored, in the presence of ATP, in membranes prepared from slices that had been labeled in the presence of fluoride. In conclusion, our data show that fluoride, at a concentration similar to that used to stimulate directly Gq/11-coupled phospholipase C, effectively blocks the synthesis of phospholipase C substrates from PtdIns.

Platelet-derived growth factor-BB (PDGF-BB) regulation of migration and focal adhesion kinase phosphorylation in rabbit aortic vascular smooth muscle cells: roles of phosphatidylinositol 3-kinase and mitogen-activated protein kinases.

Phosphatidylinositol 3'-kinase (PI3-kinase) is implicated in cell migration and focal adhesion kinase (FAK) phosphorylation. In contrast, it has been proposed that mitogen-activated protein (MAP) kinases are essential for proliferation but may be dissociated from chemotactic signalling. We investigated the roles of PI3-kinase and p42/p44 MAP kinases in cell migration and FAK tyrosine phosphorylation induced by platelet-derived growth factor-BB (PDGF-BB) in rabbit aortic vascular smooth muscle cells (VSMCs). The roles of PI3-kinase and MAP kinase pathways in the chemotactic response to insulin-like growth factor-I (IGF-I) were also examined. The roles of PI3-kinase and p42/p44 MAP kinases were assessed using the PI3-kinase inhibitors, wortmannin and LY294002, and an inhibitor of MAP kinase kinase, PD98059. PI3-kinase activity was measured by phosphatidylinositol phosphorylation in anti-phosphotyrosine immunoprecipitates and by thin layer chromatography of phosphorylated products. Phosphorylation was assessed by immunoprecipitation with anti-phosphotyrosine antibodies and Western blotting with FAK-specific antibody. Migration was evaluated in a chemotaxis chamber using polycarbonate filters with an 8-mm pore size. Neither wortmannin nor LY294002 significantly reduced PDGF-BB stimulation of FAK tyrosine phosphorylation, chemotaxis or immunofluorescent staining of focal adhesions in VSMCs. PD98059, a specific inhibitor of MAP kinase activation, did not inhibit FAK tyrosine phosphorylation but markedly inhibited the migratory response of VSMCs to PDGF-BB. IGF-I also stimulated migration of VSMCs, and, relative to the effect of PDGF-BB, induced smaller increases in PI3-kinase and MAP kinase activities. Both wortmannin and PD98059 partially inhibited the migratory response to IGF-I. PDGF-BB stimulation of both FAK tyrosine phosphorylation and migration in VSMCs are not dependent on activation of PI3-kinase. While PDGF-BB stimulation of FAK tyrosine phosphorylation is not dependent on p42/p44 MAP kinase activation, PDGF-BB and IGF-I both stimulate p42/p44 MAP kinase activity and the chemotactic response to these factors is partially dependent on MAP kinase activation.

Enhancement of phosphoinositide 3-kinase (PI 3-kinase) activity by membrane curvature and inositol-phospholipid-binding peptides.

The phosphorylation of phosphatidylinositol (PtdIns) on the 3' position of the inositol ring by phosphoinositide 3-kinase (PI 3-kinase) is shown to depend strongly on the curvature of liposomes containing a mixture of phosphatidylcholine (PtdCho) and PtdIns. Vesicles with an average diameter of 50 nm are phosphorylated 100 times faster than chemically identical vesicles with an average diameter greater than 300 nm. The low reactivity of large vesicles is not due to the difference in vesicle number for large and small vesicles at constant total lipid, nor to occlusion of lipid surfaces in multilammelar structures, and can be reversed by addition of low (< 1:100) molar ratios of either the PtdIns transfer protein sec14p or a ten-residue peptide derived from the inositol-phospholipid-binding site of gelsolin. Similar measurements using PI 4-kinase showed a weak dependence on vesicle size. The strong dependence of PI 3-kinase function on membrane curvature suggests possible localization of PI 3-kinase activity at sites where clustering of receptors, for example, may locally deform the membrane, and suggests that once PI 3-kinase is localized and activated at surface sites, the reaction may become self-accelerating.

Molecular Cloning and Functional Characterization of Murine Sphingosine Kinase

Sphingosine-1-phosphate (SPP) is a novel lipid messenger that has dual function. Intracellularly it regulates proliferation and survival, and extracellularly, it is a ligand for the G protein-coupled receptor Edg-1. Based on peptide sequences obtained from purified rat kidney sphingosine kinase, the enzyme that regulates SPP levels, we report here the cloning, identification, and characterization of the first mammalian sphingosine kinases (murine SPHK1a and SPHK1b). Sequence analysis indicates that these are novel kinases, which are not similar to other known kinases, and that they are evolutionarily conserved. Comparison with Saccharomyces cerevisiae and Caenorhabditis elegans sphingosine kinase sequences shows that several blocks are highly conserved in all of these sequences. One of these blocks contains an invariant, positively charged motif, GGKGK, which may be part of the ATP binding site. From Northern blot analysis of multiple mouse tissues, we observed that expression was highest in adult lung and spleen, with barely detectable levels in skeletal muscle and liver. Human embryonic kidney cells and NIH 3T3 fibroblasts transiently transfected with either sphingosine kinase expression vectors had marked increases (more than 100-fold) in sphingosine kinase activity. The enzyme specifically phosphorylated D-erythro-sphingosine and did not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, D,L-threo-dihydrosphingosine or N, N-dimethylsphingosine. The latter two sphingolipids were competitive inhibitors of sphingosine kinase in the transfected cells as was previously found with the purified rat kidney enzyme. Transfected cells also had a marked increase in mass levels of SPP with a concomitant decrease in levels of sphingosine and, to a lesser extent, in ceramide levels. Our data suggest that sphingosine kinase is a prototypical member of a new class of lipid kinases. Cloning of sphingosine kinase is an important step in corroborating the intracellular role of SPP as a second messenger.

Newly Synthesized Phosphatidylinositol Phosphates Are Required for Synaptic Norepinephrine but Not Glutamate or γ-Aminobutyric Acid (GABA) Release

Newly synthesized phosphatidylinositol phosphates have been implicated in many membrane-trafficking reactions. They are essential for exocytosis of norepinephrine in PC12 cells and chromaffin cells, suggesting a function in membrane fusion. We have now studied the role of phosphatidylinositol phosphates in synaptic vesicle exocytosis using synaptosomes. Under conditions where phosphorylation of phosphatidylinositols is blocked, norepinephrine secretion was nearly abolished whereas glutamate and GABA release was still elicited. Thus phosphatidylinositides are essential only for some membrane fusion reactions, and exocytotic release mechanisms differ between neurotransmitters.

An Essential Role for a Small Synaptic Vesicle-Associated Phosphatidylinositol 4-Kinase in Neurotransmitter Release

Glutamate release from nerve terminals is the consequence of Ca2+-triggered fusion of small synaptic vesicles with the presynaptic plasma membrane. ATP dependence of neurotransmitter release has been suggested to be founded, in part, on phosphorylation steps preceding membrane fusion. Here we present evidence for an essential role of phosphatidylinositol phosphorylation in stimulated release of neurotransmitter glutamate from isolated nerve terminals (synaptosomes). Specifically, we show that a phosphatidylinositol 4-kinase (PtdIns 4-kinase) activity resides on nerve terminal-derived small synaptic vesicles (SSVs) and that inhibition of the PtdIns 4-kinase activity in intact synaptosomes leads to attenuation of the evoked release of glutamate. The attenuation of transmitter release is reversible and correlates with respective changes in intrasynaptosomal PtdIns 4-kinase activity. Because only the Ca2+-dependent release of glutamate is affected, regulation appears to be at the level of exocytosis. Taken together, our data imply a mandatory role for PtdIns 4-kinase and phosphoinositide products in the regulated exocytosis of SSV in mammalian nerve terminals.

Increased levels of phosphatidylinositol 3-kinase activity in colorectal tumors.

Phosphatidylinositol 3-kinase (PI 3-kinase), an enzyme that phosphorylates inositol phospholipids at the D-3 position of the inositol ring, has been implicated in the signaling pathways regulating cell growth by virtue of its activation in response to various mitogenic stimuli. In spite of the considerable attention PI 3-kinase has received with regard to its possible role in the mitogenic pathways in hematopoietic malignancies, there are few reports of investigations into PI 3-kinase activity in solid tumors. Colorectal tumor tissue and normal-appearing colonic mucosa from the same patients were homogenized and solubilized and adjusted to equal protein levels. PI 3-kinase then was immunoprecipitated from 200 microg of the solubilized tissue using a polyclonal antibody to the p85 subunit of PI 3-kinase. PI 3-kinase activity was assessed using phosphatidylinositol as the substrate and the assay product analyzed by thin-layer chromatography. Phosphorylation of phosphatidylinositol in the D-3 position was confirmed by high performance liquid chromatography analysis of deacylated and deglycerated products. Thirty-two of the 37 tumors tested (86%) demonstrated increased PI 3-kinase activity compared with normal-appearing mucosa from the same patients (overall mean increase+/-standard error of the mean=3.8+/-0.6-fold; P < 0.05, Student's t test for paired data). The frequency and extent of increased PI 3-kinase enzyme activity in tumors did not correlate with clinical parameters or the presence of oncogenic ras mutations. In this study colorectal tumors exhibited enhanced PI 3-kinase activity compared with normal colonic mucosa, raising the possibility that PI 3-kinase may be a potential target for new strategies for the treatment of colorectal carcinoma.

Purification and Characterization of Rat Kidney Sphingosine Kinase

Sphingosine kinase catalyzes the formation of the bioactive sphingolipid metabolite sphingosine 1-phosphate, which plays important roles in numerous physiological processes, including growth, survival, and motility. We have purified rat kidney sphingosine kinase 6 x 10(5)-fold to apparent homogeneity. The purification procedure involved ammonium sulfate precipitation followed by chromatography on an anion exchange column. Partially purified sphingosine kinase was found to be stabilized by the presence of high salt, and thus, a scheme was developed to purify sphingosine kinase using sequential dye-ligand chromatography steps (since the enzyme bound to these matrices even in the presence of salt) followed by EAH-Sepharose chromatography. This 385-fold purified sphingosine kinase bound tightly to calmodulin-Sepharose and could be eluted in high yield with EGTA in the presence of 1 M NaCl. After concentration, the calmodulin eluate was further purified by successive high pressure liquid chromatography separations on hydroxylapatite, Mono Q, and Superdex 75 gel filtration columns. Purified sphingosine kinase has an apparent molecular mass of approximately 49 kDa under denaturing conditions on SDS-polyacrylamide gel, which is similar to the molecular mass determined by gel filtration, suggesting that the active form is a monomer. Sphingosine kinase shows substrate specificity for D-erythro-sphingosine and does not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, DL-threo-dihydrosphingosine, or N,N-dimethylsphingosine. However, the latter two sphingolipids were potent competitive inhibitors. With sphingosine as substrate, the enzyme had a broad pH optimum of 6.6-7.5 and showed Michaelis-Menten kinetics, with Km values of 5 and 93 microM for sphingosine and ATP, respectively. This study provides the basis for molecular characterization of a key enzyme in sphingolipid signaling.

A novel phosphorylated lipid counteracts activation of the renal plasma membrane (Ca2+ + Mg2+)ATPase by endogenous phosphatidylinositol-4-phosphate.

The plasma membrane (Ca2+ + Mg2+)ATPase is activated by acidic phospholipids in reconstituted systems. In this report it is shown that reversible phosphorylation of endogenous phosphatidylinositol regulates the renal plasma membrane (Ca2+ + Mg2+)ATPase, and that a novel phosphorylated lipid that can be isolated from the same membrane strongly counteracts the stimulatory effect of phosphatidylinositol-4-phosphate.

Prolactin induced tyrosine phosphorylation of p59fyn may mediate phosphatidylinositol 3-kinase activation in Nb2 cells.

Our previous studies indicated that PI3-kinase is involved in prolactin (PRL) signalling. We have now examined the involvement of the src tyrosine kinase, fyn, in PRL-induced the activation of PI3-kinase in the rat lymphoma cell line, Nb2. Cells were stimulated with increasing doses of PRL, lysed and immunoprecipitated with anti-fyn specific antibody. Then PI3-kinase activity was measured as the increase in the phosphorylation of phosphatidylinositol to phosphatidylinositol 3-phosphate separated by TLC. Our data indicated that, in PRL treated cells, co-precipitation of PI3-kinase with anti-fyn antiserum led to time and dose-dependent activation of PI3-kinase in vitro and that this activation was blocked by the addition of LY294002. However, LY294002 appeared to have no effect on fyn autophosphorylation. Furthermore, the physical association of PI3-kinase with fyn was confirmed by Western blot analysis employing the same specific antisera. These data provide evidence that PRL-induced activation of PI3-kinase may be mediated by the tyrosine phosphorylation of fyn in Nb2 cells.

Calmodulin Activates Phosphatidylinositol 3-Kinase

Calmodulin and phosphatidylinositol 3-kinase are vital components of a number of common intracellular events. Calmodulin, a ubiquitous Ca2+-dependent effector protein, regulates multiple processes in eukaryotic cells, including cytoskeletal organization, vesicular trafficking, and mitogenesis. Phosphatidylinositol 3-kinase participates in events downstream of the receptors for insulin and other growth factors. Here we demonstrate by coimmunoprecipitation and affinity chromatography that Ca2+/calmodulin associates with Src homology 2 domains in the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase, thereby significantly enhancing phosphatidylinositol 3-kinase activity in vitro and in intact cells. Furthermore, CGS9343B, a calmodulin antagonist, inhibited basal and Ca2+-stimulated phosphorylation of phosphatidylinositol in intact cells. These data demonstrate a novel mechanism for modulating phosphatidylinositol 3-kinase and provide a direct link between components of two fundamental signaling pathways.

Phosphoinositides as spatial regulators of membrane traffic

Inositol phospholipids are a focus of renewed interest with the discovery of their unanticipated pivotal roles in membrane trafficking events. Reversible phosphorylation of phosphatidylinositol generates spatially localized signals on membranes that recruit or activate proteins essential for cell membrane budding, fission and fusion. Recent advances have taken place in the characterization of lipid kinases and phosphoinositide-regulated effector proteins, and in the elucidation of phospholipase D mediated mechanisms involving ADP ribosylation factor and Rho family proteins. The roles played by phosphoinositides in aspects of secretory granule formation, fusion and endocytosis indicate the importance of phosphorylated lipids for neurotransmitter release.

Regulation of osteoclast function.

Regulation of osteoclast function.

Extension of long cellular processes of hepatic stellate cells cultured on extracellular type I collagen gel by microtubule assembly: observation utilizing time-lapse video-microscopy.

Hepatic stellate cells cultured on or in freshly prepared type I collagen gel as a substratum were induced to elongate long cellular processes. The extension of the cellular processes was monitored by using video-enhanced optical microscopy. The cellular processes seemed to extend along the extracellular type I collagen fibers. Once extended cellular processes after overnight culture on type I collagen gel were retracted by cytoskeleton degradation with colchicine or cytochalasin B. The cellular processes were also retracted by treatment with protein kinase inhibitor, herbimycin A or staurosporin, or with phosphatidylinositol 3-kinase inhibitor, wortmannin. The effects of colchicine, herbimycin A, staurosporin, or wortmannin were drastic, and the cells were finally changed to a round shape within a few hours, as seen also after cold-treatment at 4 degrees C. Cytochalasin B also time-dependently retracted the extended cellular processes. These results indicated that the cultured stellate cells were induced to elongate cellular processes by cell surface binding to type I collagen fibrils, followed by protein or phosphatidylinositol phosphorylation and finally F-actin and microtubule assembly. Extended long cellular processes seem to reflect the in vivo structure of hepatic stellate cells, and molecular mechanism for the extension and maintenance of cellular processes was proposed.

Phosphopleckstrin Inhibits Gβγ-activable Platelet Phosphatidylinositol-4,5-bisphosphate 3-Kinase

Pleckstrin, the prototypic protein containing two copies of the pleckstrin homology domain, is a prominent substrate of protein kinase C in platelets and neutrophils. Both cell types have p85 subunit-containing phosphoinositide 3-kinase (p85/PI3K) and non-p85-containing PI3K (PI3Kgamma) that is activated by betagamma subunits of heterotrimeric GTP-binding proteins. We have shown that a PI3K product, phosphatidylinositol (PI) 3,4,5-trisphosphate, promotes pleckstrin phosphorylation in platelets. Since pleckstrin homology domains are thought to interact with Gbetagamma heterodimers and/or PI(4,5)P2, we have examined the effects of recombinant pleckstrins on platelet PI3Kgamma and p85/PI3K activities. Depending upon its phosphorylation/charged state, pleckstrin inhibits PI3Kgamma, but not p85/PI3K. Pleckstrin-mediated inhibition of PI3Kgamma is overcome by excess Gbetagamma and is restricted to PI(4,5)P2 as substrate, i.e. pleckstrin does not inhibit phosphorylation of PI()P or PI. Consistent with this, activation of protein kinase C by exposure of platelets to beta-phorbol diester (to increase endogenous pleckstrin phosphorylation) prior to platelet lysis causes inhibition of Gbetagamma-stimulatable PI3K activity only with respect to PI(4,5)P2 substrate. This phosphopleckstrin-mediated inhibition is overcome by increasing concentrations of Gbetagamma. We propose that phosphorylation of pleckstrin may constitute an important inhibitory mechanism for PI3Kgamma-mediated cell signaling.

CD28-mediated cytotoxicity by the human leukemic NK cell line YT involves tyrosine phosphorylation, activation of phosphatidylinositol 3-kinase, and protein kinase C.

The human leukemic cell line YT displays spontaneous cytotoxicity against CD80+ and/or CD86+ and ICAM-1+ target cells. In this work, we report that CD28-mediated cytotoxicity of YT involves tyrosine phosphorylation and activation of phosphatidylinositol (PI) 3-kinase, the Tec kinase Itk/Emt, and protein kinase C (PKC). YT mediates lysis of CD80+/CD86+ B lymphoblastoid cell lines and the murine mastocytoma p815 transfected with CD80 or CD86. The lysis was inhibited by two different Pi 3-kinase inhibitors, wortmannin and LY294002. The PKC inhibitors calphostin C and bisindolylmaleimide GF109203X also abolished YT-mediated cytotoxicity. Furthermore, exocytosis of cytolytic effector molecules was also inhibited by PI 3-kinase inhibitors and PKC inhibitors. PMA together with Ionomycin did not induce granule exocytosis or cytotoxicity by YT cells. Treatment of YT cells with PMA for up to 20 h, which depleted PMA-responsive PKC isoforms, had no effect on the CD28-mediated cytotoxicity. This cytotoxicity displayed by PMA-treated YT cells, however, could still be inhibited by Pi 3-kinase inhibitors and PKC inhibitors. Taken together, these results are consistent with a model in which activation of CD28 and LFA-1 induces tyrosine phosphorylation of the CD28 cytoplasmic domain, recruitment and activation of PI 3-kinase, as well as the Tec kinase Itk/Emt, and the activation of PMA-nonresponsive PKC isoenzymes. Activation of PI 3-kinase and PMA-nonresponsive PKC isoenzymes is shown to be involved directly in cytolytic granule release by YT cells.

Chromaffin granule-associated phosphatidylinositol 4-kinase activity is required for stimulated secretion.

Permeabilized bovine adrenal chromaffin cells have been used to characterize the MgATP requirement of processes preceding exocytosis. Incubation of primary cultures with the membrane-permeable phenylarsine oxide (PAO) at 20 microM inhibited the phosphorylation of phosphatidylinositol (PtdIns) and completely blocked secretion. This block could be reversed by addition of 2,3-dimercaptopropanol to the permeabilized cells. Simultaneous addition of [gamma32P]ATP and 2,3-dimercaptopropanol permitted a comparison between recovery of secretion and phosphorylation of intracellular components. Recovery of secretion closely correlated with phosphorylation of PtdIns and PtdIns4P. Subcellular fractionation of permeabilized cells after recovery of secretion revealed that the majority of newly phosphorylated PtdIns4P was localized on the chromaffin granules. In accordance with these results, PtdIns 4-kinase activity was found in protein extracts of permeabilized cells as well as associated with purified chromaffin granules, sensitive in both cases to PAO. Additionally, PtdIns 4-kinase activity in these two assays was inhibited by quercetin. In permeabilized cells, quercetin decreased the levels of labeled PtdIns4P and Ptdlns(4,5)P2 and inhibited secretion. Our data suggest that a chromaffin granule-associated PtdIns 4-kinase acts in the priming of exocytosis.

Relationship between type II phosphatidylinositol 4-kinase activity and protein tyrosine phosphorylation in membranes from normal and sickle red cells.

To assess the origin of the previously reported higher type II phosphatidylinositol 4-kinase (PtdIns 4-kinase) activity of sickle-red-cell membranes [Rhoda-Hardy-Dessources, M.D., de Neef, R.S., Mérault, G.& Giraud, F. (1993) Biochim. Biophs. Acta 1181, 90-96], we have investigated the possible involvement of protein kinase C and tyrosine kinases in the regulation of the lipid kinase activity. Both protein kinase activities were found to be markedly higher in membranes from the pathological cells. When isolated normal-red-cell or sickle-red-cell membranes were assayed, phosphatidylinositol phosphorylation activity was not significantly modified after phorbol ester modulation of protein kinase C. In contrast, stimulation (with sodium orthovanadate) or inhibiton (by tyrphostin) of tyrosine phosphorylation led respectively, to increased or decreased PtdIns 4-kinase activity in membranes from both cell types. Moreover, immunoprecipitations of membrane extracts from normal and sickle red cells types with anti-PtdIns 4-kinase antibody 4C5G, followed by immunoblotting with an anti-phosphotyrosine Ig, revealed a 56-kDa band migrating with PtdIns 4-kinase activity. Taken together, these findings indicate that PtdIns 4-Kinase in red blood cells is a phosphotyrosine-containing protein and could be regulated by a mechanism involving tyrosine phosphorylation, and the increase in PtdIns 4-Kinase activity of sickle-red-cell membranes is at least in part mediated by their intrinsic tyrosine kinase activity.