Altered signal transduction in erbB-transformed cells. Implication of enhanced inositol phospholipid metabolism in erbB-induced transformation

To clarify the signal transduction mechanism of the erbB gene (virus oncogene) products leading to cell growth and transformation, the alteration of signal transduction induced by enhanced inositol phospholipid metabolism was studied in chick embryo fibroblast cells (CEF cells) transformed by gag-fused erbB gene-carrying virus (GEV cells). The incorporations of 32P into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate were markedly increased in GEV cells. In GEV cells, the activities of lipid kinases such as phosphatidylinositol (PI), PIP, and diacylglycerol (DG) kinases were also increased. The activities of other important enzymes involved in inositol phospholipid metabolism, such as CDP-DG:myo-inositol transferase and phospholipase C, were not changed in GEV cells. Increased inositol phospholipid metabolism might lead to the production of second messengers, such as 1,2-DG and inositol 1,4,5-trisphosphate. Indeed, the 1,2-DG content was also increased in GEV cells. Moreover, the activity of protein kinase C (the Ca2+/phospholipid-dependent enzyme), which should be stimulated by 1,2-DG, was elevated in GEV cells; the protein kinase C activity in the membrane fraction of GEV cells was especially high. When CEF cells were treated with tetradecanoylphorbol acetate, protein kinase C activator, plus Ca2+ ionophore, [3H]thymidine incorporation was markedly stimulated, and maximal stimulation was observed with 1 nM Ca2+ ionophore A23187 plus 100 nM TPA. On the other hand, when GEV cells were treated with TPA plus Ca2+ ionophore A23187, [3H]thymidine incorporation was consistently inhibited. Next, studies were made to determine whether the erbB gene product itself had kinase activity on PI, PIP, and DG after membranes were mildly solubilized with Triton X-100 to prevent inactivation of these kinases. Immunoprecipitates of a GEV cell lysate with antisera that reacted with the erbB gene product had PI kinase activity, whereas no activity was detected in those of lysates of uninfected CEF cells. However, the activity was very weak compared with the total cellular activity. No difference in the PIP and DG kinase activities of immunoprecipitates of cell lysates of uninfected CEF cells and GEV cells was observed. These results suggest that the erbB gene product enhances inositol phospholipid metabolism and subsequent signal transduction, but that the erbB gene product is not involved directly in lipid kinases, although it is closely associated with lipid kinase.     

Activation of protein kinase C in rat basophilic leukemia cells stimulates increased production of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate: correlation with actin polymerization.

Cross-linking of the immunoglobulin E receptor on rat basophilic leukemia (RBL)1 cells by multivalent antigen activates phosphatidylinositol (PI) kinase and phosphatidylinositol 4-phosphate (PIP) kinase leading to the increased production of PIP and phosphatidylinositol 4,5-bisphosphate (PIP2). Activators of protein kinase C (PKC), such as phorbol myristate acetate (PMA) and the synthetic diacylglycerol, 1,2-dioctanoyl-sn-glycerol (diC8), were found to have the same effect even though PMA and diC8 do not cause the activation of phospholipase C. Although the kinetics are different depending on the stimulant, activation of PKC using multivalent antigen, PMA or diC8 also causes the polymerization of actin and an increase in the F-actin content of the cells. In all cases, a good correlation was observed between F-actin levels, activation of PI and PIP kinases, and the increased production of PIP and PIP2. However, in the case of antigen, there is no correlation between actin polymerization and the total amount of PIP and PIP2. Staurosporine, an inhibitor of protein kinases, blocks the F-actin response and the increased synthesis of PIP and PIP2 with similar dose dependencies. Furthermore, depletion of PKC activity through long-term exposure to PMA, inhibited both the F-actin response and the increased synthesis of PIP and PIP2 induced by either DNP-BSA or diC8. These results suggest that activation of PKC precedes the activation of PI and PIP kinases and that under certain circumstances activation of the kinases and the increased synthesis of PIP and PIP2 may be involved in the polymerization of actin in RBL cells, possibly through the interaction of the polyphosphoinositides with actin-binding proteins such as gelsolin and profilin.     

Phosphatidylinositol-4,5-bisphosphate may antecede diacylglycerol as activator of protein kinase C

Phosphatidylserine/calcium-dependent protein kinase C (PKC) from rat brain is activated fifty times more efficiently by phosphatidylinositol-4,5-bisphosphate (PIP2) (Kapp = 0.04 mole% in Triton-lipid micelles) than by diacylglycerol (DG) (Kapp = 2 mole%). Both effector lipids appear to bind to the same site but PIP2 may confer a narrower substrate specificity on the kinase. DG, which together with inositol trisphosphate (IP3) is generated by hydrolysis from PIP2 after cell stimulation, has been considered the natural activator of the kinase but it is likely to be anteceded in this function by PIP2; DG may perhaps retain the function of a back-up activator. The lack of PKC-activation by phosphatidylinositol (PI) or phosphatidylinositol-4-phosphate (PIP) opens the possibility that the Inositide Shuttle, PI reversible PIP reversible PIP2, has a role in controlling the activity of the kinase.     

Norepinephrine and endothelin activate diacylglycerol kinases in caveolae/rafts of rat mesenteric arteries: agonist-specific role of PI3-kinase

The phosphatidylinositol (PI) signaling pathway mediates norepinephrine (NE)- and endothelin-1 (ET-1)-stimulated vascular smooth muscle contraction through an inositol-trisphosphate-induced rise in intracellular calcium and diacylglycerol (DG) activation of protein kinase C (PKC). Subsequent activation of DG kinases (DGKs) metabolizes DG to phosphatidic acid (PA), potentially regulating PKC activity. Because precise regulation and spatial restriction of the PI pathway is necessary for specificity, we have investigated whether this occurs within caveolae/rafts, specialized plasma membrane microdomains implicated in vascular smooth muscle contraction. We show that components of the PI signaling cascade-phosphatidylinositol 4,5-bisphosphate (PIP(2)), PA, and DGK-theta are present in caveolae/rafts prepared from rat mesenteric small arteries. Stimulation with NE or ET-1 induced [(33)P]PIP(2) hydrolysis solely within caveolae/rafts. NE stimulated an increase in DGK activity in caveolae/rafts alone, whereas ET-1 activated DGK in caveolae/rafts and noncaveolae/rafts; however, [(33)P]PA increased in all fractions with both agonists. Previously, we reported that NE activated DGK-theta in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner; here, we describe PI3-kinase-dependent DGK activation and [(33)P]PA production in caveolae/rafts in response to NE but not ET-1. Additionally, PKB, a potential activator of DGK-theta, translocated to caveolae/rafts in response to NE but not ET-1, and PI3-kinase inhibition prevented this. Furthermore, PI3-kinase inhibition reduced the sensitivity of contraction to NE but not ET-1. Our study shows that caveolae/rafts are major sites of vasoconstrictor hormone activation of the PI pathway in intact small arteries and suggest a link between lipid signaling events within caveolae/rafts and contraction.     

Epidermal growth factor stimulates diacylglycerol kinase in isolated plasma membrane vesicles from A431 cells

We have examined the effect of epidermal growth factor(EGF) on three kinds of kinases activities, phosphatidylinositol(PI) kinase, phosphatidylinositol 4-phosphate[PI(4)P] kinase and diacylglycerol(DG) kinase that make important roles in the regulation of inositol phospholipids metabolism. When isolated plasma membrane vesicles from A431 cells were incubated at 30 degrees C with [gamma-32P]ATP and exogenously added DG, EGF enhanced the activity of DG kinase approximately 2-fold. This stimulation is found to be dose-dependent with a half maximal activation at 1 nM. In this case, EGF increased Vmax without changing Km Value for ATP or DG. Although this activation was observed in the absence of detergent, it was more evident when membrane vesicles were treated with 1 mM deoxycholate. Interestingly, the effect of EGF was only detected in magnesium containing medium. The use of manganese instead of magnesium diminished the stimulatory effect in either condition, presence or absence of deoxycholate. On the other hand, the stimulation of PI kinase or PI(4)P kinase activity was not caused by EGF. These results suggest that DG kinase activation by EGF makes important roles in cellular responses leading to cell growth.     

Ca2+ regulation of phosphatidylinositol turnover in the plasma membrane of tobacco suspension culture cells.

The biochemical properties of the enzymes involved in phosphatidylinositol (PI) turnover in higher plants were investigated using the plasma membrane isolated from tobacco suspension culture cells by aqueous two-phase partitioning. Submicromolar concentrations of Ca2+ inhibited PI kinase and phosphatidylinositol 4-phosphate (PIP) kinase and stimulated phospholipase C. Diacylglycerol (DG) kinase was inhibited by Ca2+, but required a higher concentration than the physiological level. From the above results we postulate the following scheme: signal coupled activation of phospholipase C produces IP3 which induces Ca2+ release from the intracellular Ca2+ compartment, the increased cytoplasmic Ca2+ in turn activates phospholipase C and causes a further increase of the cytoplasmic Ca2+ level. This inhibits PI kinase and PIP kinase and brings about a limited supply of PIP2, the substrate of phospholipase C. Consequently, IP3 production decreases and Ca2+ mobilization ceases. Then cytosolic Ca2+ returns to the stationary level by the Ca2+ pump at the plasma membrane and at the endoplasmic reticulum and Ca2+/H+ antiporter at the plasma membrane and at the tonoplast.     

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.     

Regulation of polyphosphoinositide synthesis in cardiac membranes

The relative distribution of phosphatidylinositol (PI) and phosphatidylinositol-4-phosphate (PIP) kinase activities in enriched cardiac sarcolemma (SL), sarcoplasmic reticulum (SR), and mitochondrial fractions was investigated. PI and PIP kinase activities were assayed by measuring 32P incorporation into PIP and phosphatidylinositol 4,5-bisphosphate (PIP2) from endogenous and exogenous PI in the presence of [gamma-32P]ATP. PI and PIP kinase activities were present in SL, SR, and mitochondrial fractions prepared from atria and ventricles although the highest activities were found in SL. A similar membrane distribution was found for PI kinase activity measured in the presence of detergent and exogenous PI. PI and PIP kinase activities were detectable in the cytosol providing exogenous PI and PIP and Triton X-100 were present. Further studies focused on characterizing the properties and regulation of PI and PIP kinase activities in ventricular SL. Alamethacin, a membrane permeabilizing antibiotic, increased 32P incorporation into PIP and PIP2 4-fold. PI and PIP kinase activities were Mg2+ dependent and plateaued within 15-20 min at 25 degrees C. Exogenous PIP and PIP2 (0.1 mM) had no effect on PIP and PIP2 labeling in SL in the absence of Triton X-100 but inhibited PI kinase activity in the presence of exogenous PI and Triton X-100. Apparent Km's of ATP for PI and PIP kinase were 133 and 57 microM, respectively. Neomycin increased PIP kinase activity 2- to 3-fold with minor effects on PI kinase activity. Calmidazolium and trifluoperazine activated PI kinase activity 5- to 20-fold and completely inhibited PIP kinase activity. Quercetin inhibited PIP kinase 66% without affecting PI kinase activity. NaF and guanosine 5'-O-(3-thiotriphosphate) had no effect on PI and PIP kinase activities, indicating that these enzymes were not modulated by G proteins. The probability that PIP and PIP2 synthesis in cardiac sarcolemma is regulated by product inhibition and phospholipase C was discussed.     

Sustained diacylglycerol formation from inositol phospholipids in angiotensin II-stimulated vascular smooth muscle cells

Angiotensin II acts on cultured rat aortic vascular smooth muscle cells to stimulate phospholipase C-mediated hydrolysis of membrane phosphoinositides and subsequent formation of diacylglycerol and inositol phosphates. In intact cells, angiotensin II induces a dose-dependent increase in diglyceride which is detectable after 5 s and sustained for at least 20 min. Angiotensin II (100 nM)-stimulated diglyceride formation is biphasic, peaking at 15 s (227 +/- 19% control) and at 5 min (303 +/- 23% control). Simultaneous analysis of labeled inositol phospholipids shows that at 15 s phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP) decline to 52 +/- 6% control and 63 +/- 5% control, respectively, while phosphatidylinositol (PI) remains unchanged. In contrast, at 5 min, PIP2 and PIP have returned toward control levels (92 +/- 2 and 82 +/- 4% control, respectively), while PI has decreased substantially (81 +/- 2% control). The calcium ionophore ionomycin (15 microM) stimulates diglyceride accumulation but does not cause PI hydrolysis. 4 beta-Phorbol 12-myristate 13-acetate, an activator of protein kinase C, inhibits early PIP and PIP2 breakdown and diglyceride formation, without inhibiting late-phase diglyceride accumulation. Thus, angiotensin II induces rapid transient breakdown of PIP and PIP2 and delayed hydrolysis of PI. The rapid attenuation of polyphosphoinositide breakdown is likely caused by a protein kinase C-mediated inhibition of PIP and PIP2 hydrolysis. While in vascular smooth muscle stimulated with angiotensin II inositol 1,4,5-trisphosphate formation is transient, diglyceride production is biphasic, suggesting that initial and sustained diglyceride formation from the phosphoinositides results from different biochemical and/or cellular processes.     

Stimulation of polyphosphoinositide turnover upon activation of protein kinases in human erythrocytes

Activation of protein kinase C in erythrocytes by 4-beta-phorbol 12-myristate 13-acetate (PMA) resulted in a parallel stimulation (time course and dose response) of the phosphorylation of both membrane proteins (heterodimers of 107 kDa and 97 kDa, protein 4.1 and 4.9, respectively) and of phosphatidylinositol 4-phosphate (PIP) and, to a lesser extent, of phosphatidylinositol 4,5-bisphosphate (PIP2). Evidence that the effect on lipid was mediated by protein kinase C activation and not by a direct action of PMA was provided by (1) the lack of effect of a phorbol ester that did not activate protein kinase C or of PMA addition on isolated membranes from control erythrocytes, (2) the reversal of the effect in the presence of protein kinase C inhibitors (alpha-cobrotoxin, H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine) or trifluoperazine). PMA treatment did not change the specific activity of ATP or the content of PIP2, but increased the content of PIP and decreased that of PI, indicating that the phosphorylation or dephosphorylation reactions linking PI and PIP were the target for the action of PMA. PMA treatment had no effect on the Ca2+-dependent PIP/PIP2 phospholipase C activity measured in isolated membranes. Mezerein, another protein kinase activator, had similar effects on both protein and lipid phosphorylation, when added with alpha-cobrotoxin. Activation of protein kinase A by cAMP also produced increases in phosphorylation, although quantitatively different from those induced by protein kinase C, in proteins and PIP. Simultaneous addition of PMA and cAMP at maximal doses resulted in only a partially additive effect on PIP labelling. These results show that inositol lipid turnover can be modulated by a protein kinase C and protein kinase A-dependent process involving the phosphorylation of a common protein. This could be PI kinase or PIP phosphatase or another protein regulating the activity of these enzymes.     

Effect of transforming growth factor-alpha on inositol phospholipid metabolism in human epidermoid carcinoma cells

Transforming growth factor-alpha (TGF-alpha) stimulates (in a dose-dependent manner) the incorporation of [32P]Pi into phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA) in the human epidermoid carcinoma cell line (A431). The effect of TGF-alpha on the incorporation was found to be similar to that of EGF. On the other hand, a striking difference in the activation of diacylglycerol (DG) kinase activity was seen between TGF-alpha and EGF. At least 100 times more TGF-alpha was required to achieve maximal stimulation of DG kinase activity relative to EGF. These results suggest that the activation of DG kinase by TGF-alpha may involve a mechanism independent from or subsequent to activation of the EGF receptor.     

Phosphatidylinositol kinase activities in normal and Rous sarcoma virus-transformed cells.

The phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and diacylglycerol kinase activities in the plasma membrane-rich fraction of chicken embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus increased when the cells were shifted from the nonpermissive temperature, 41 degrees C, to the permissive temperature, 35 degrees C. Temperature shift from 35 to 41 degrees C decreased the lipid kinase activities in the membrane vesicles. These changes accompanied the changes observed in pp60v-src protein kinase activity. Thermal inactivation at 41 degrees C did not appreciably reduce PI and PIP kinase activities in membrane vesicles prepared from uninfected or Rous sarcoma virus-transformed cells, whereas pp60v-src protein kinase activity in the membrane vesicles was rapidly inactivated under the same conditions. These data suggest that pp60v-src may indirectly enhance PI and PIP phosphorylation but not directly contribute to this pathway.     

The de novo phospholipid effect of insulin is associated with increases in diacylglycerol, but not inositol phosphates or cytosolic Ca2+.

We have previously reported that insulin increases the synthesis de novo of phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG) in BC3H-1 myocytes and/or rat adipose tissue. Here we have further characterized these effects of insulin and examined whether there are concomitant changes in inositol phosphate generation and Ca2+ mobilization. We found that insulin provoked very rapid increases in PI content (20% within 15 s in myocytes) and, after a slight lag, PIP and PIP2 content in both BC3H-1 myocytes and rat fat pads (measured by increases in 32P or 3H content after prelabelling phospholipids to constant specific radioactivity by prior incubation with 32Pi or [3H]inositol). Insulin also increased 32Pi incorporation into these phospholipids when 32Pi was added either simultaneously with insulin or 1 h after insulin. Thus, the insulin-induced increase in phospholipid content appeared to be due to an increase in phospholipid synthesis, which was maintained for at least 2 h. Insulin increased DAG content in BC3H-1 myocytes and adipose tissue, but failed to increase the levels of inositol monophosphate (IP), inositol bisphosphate (IP2) or inositol trisphosphate (IP3). The failure to observe an increase in IP3 (a postulated 'second messenger' which mobilizes intracellular Ca2+) was paralleled by a failure to observe an insulin-induced increase in the cytosolic concentration of Ca2+ in BC3H-1 myocytes as measured by Quin 2 fluorescence. Like insulin, the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the transport of 2-deoxyglucose and aminoisobutyric acid in BC3H-1 myocytes. These effects of insulin and TPA appeared to be independent of extracellular Ca2+. We conclude that the phospholipid synthesis de novo effect of insulin is provoked very rapidly, and is attended by increases in DAG but not IP3 or Ca2+ mobilization. The insulin-induced increase in DAG does not appear to be a consequence of phospholipase C acting upon the expanded PI + PIP + PIP2 pool, but may be derived directly from PA. Our findings suggest the possibility that DAG (through protein kinase C activation) may function as an important intracellular 'messenger' for controlling metabolic processes during insulin action.     

Polyphosphoinositide phospholipase C in wheat root plasma membranes. Partial purification and characterization.

The effect of various detergents on polyphosphoinositide-specific phospholipase C activity in highly purified wheat root plasma membrane vesicles was examined. The plasma membrane-bound enzyme was solubilized in octylglucoside and purified 25-fold by hydroxylapatite and ion-exchange chromatography. The purified enzyme catalyzed the hydrolysis of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) with specific activities of 5 and 10 mumol/min per mg protein, respectively. Phosphatidylinositol (PI) was not a substrate. Optimum activity was between pH 6-7 (PIP) and pH 6-6.5 (PIP2). The enzyme was dependent on micromolar concentrations of Ca2+ for activity, and millimolar Mg2+ further increased the activity. Other divalent cations (4 mM Ca2+, Mn2+ and Co2+) inhibited (PIP2 as substrate) or enhanced (PIP as substrate) phospholipase C activity.     

Protein kinase C activity in platelets from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY)

Calcium-activated phospholipid dependent protein kinase (protein kinase C) activity in platelets was measured in 4, 12, and 20-week-old SHR and WKY. At age 4-weeks, there was no significant difference in protein kinase C activity and systolic blood pressure between SHR and WKY. In 12 and 20-week-old SHR, both protein kinase C activity and systolic blood pressure were significantly higher than in the age-matched WKY. These results suggest that protein kinase C may be involved in the control of blood pressure in SHR and WKY.     

Regulation of type IIalpha phosphatidylinositol phosphate kinase localisation by the protein kinase CK2.

Inositol lipid synthesis is regulated by several distinct families of enzymes [1]. Members of one of these families, the type II phosphatidylinositol phosphate kinases (PIP kinases), are 4-kinases and are thought to catalyse a minor route of synthesis of the multifunctional phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) from the inositide PI(5)P [2]. Here, we demonstrate the partial purification of a protein kinase that phosphorylates the type IIalpha PIP kinase at a single site unique to that isoform - Ser304. This kinase was identified as protein kinase CK2 (formerly casein kinase 2). Mutation of Ser304 to aspartate to mimic its phosphorylation had no effect on PIP kinase activity, but promoted both redistribution of the green fluorescent protein (GFP)-tagged enzyme in HeLa cells from the cytosol to the plasma membrane, and membrane ruffling. This effect was mimicked by mutation of Ser304 to alanine, although not to threonine, suggesting a mechanism involving the unmasking of a latent membrane localisation sequence in response to phosphorylation.     

Kinetic analysis of guanosine 5'-O-(3-thiotriphosphate) effects on phosphatidylinositol turnover in NRK cell homogenates

Addition of the guanine nucleotide analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to [3H]inositol-labeled NRK cell homogenates resulted in rapid breakdown of cellular polyphosphoinositides. GTP gamma S stimulated phospholipase C, resulting in a more than 4-fold increase in the hydrolysis rates of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bis(phosphate) (PIP2). No significant effect of GTP gamma S on direct phosphatidylinositol (PI) hydrolysis was detected. There was an increase in water-soluble inositols, with inositol tris(phosphate) (IP3) levels increasing at least 10 times over the decrease seen in PIP2, indicating that PIP kinase activity was also accelerated following GTP gamma S addition. Inositol 1,4,5-tris(phosphate) peaked rapidly after GTP gamma S addition (less than 2 min) while inositol 1,3,4-tris-(phosphate) was produced more slowly and leveled off after approximately 10 min. The differential equations describing conversion between intermediates in the PI turnover pathway were solved and fitted to data obtained from both [3H]inositol and [32P]phosphate fluxes by nonlinear least-squares analysis. GTP gamma S effects on the pseudo-first-order rate constants for the lipase, kinase, and phosphatase steps were determined from the analysis. From these measurements it can be estimated that, in the presence of GTP gamma S and calcium buffered to 130 nM, hydrolysis of PIP2 accounts for at least 10 times as much diacylglycerol as direct PI breakdown despite the 100-fold excess of PI over PIP2. From the kinetic model it is predicted that small changes in the activities of PI and PIP kinases can have large but different effects on the level of IP3 and diacylglycerol following GTP gamma S addition. These results argue that regulation of PI and PIP kinases may be important for determining both cellular IP3 and diacylglycerol levels.     

B cell activation. VI. Effects of exogenous diglyceride and modulators of phospholipid metabolism suggest a central role for diacylglycerol generation in transmembrane signaling by mIg

Previous evidence indicates that in vitro activators of protein kinase C, such as phorbol myristate acetate (PMA), are able to induce early activation events in murine B cells, including membrane depolarization and increased I-A antigen expression. These same events are induced by specific antigen and anti-receptor antibody. This evidence suggests that protein kinase C activation may be an important intermediary event in mIg-mediated transmembrane signaling. Previously, investigators have suggested that protein kinase C activation is regulated by a novel second messenger, diacylglycerol (DG), and DG is generated by phosphatidylinositol (PI) hydrolysis after receptor-ligand interaction in many systems. In view of this concept, we examined the effects of nonspecific activators and inhibitors of DG production and DG itself on membrane potential and levels of I-A antigen expression in murine B cells. Our results indicate that exposure to DG, or induction of DG production by treatment of B cells with exogenous phospholipase C, results in depolarization and increased I-A antigen expression similar to that induced by anti-receptor antibody and specific antigen. Furthermore, we demonstrate that depolarization and increased I-A expression induced by anti-receptor antibody is blocked under conditions in which DG production is inhibited. As expected, based on its direct activation of protein kinase C, PMA stimulation is unaffected by this inhibition. These results support our earlier hypothesis that occupancy of antigen receptors on B cells is linked to subsequent activation events by PI hydrolysis, DG generation, and protein kinase C activation.     

Regulation of type IIα phosphatidylinositol phosphate kinase localisation by the protein kinase CK2

Inositol lipid synthesis is regulated by several distinct families of enzymes [1]. Members of one of these families, the type II phosphatidylinositol phosphate kinases (PIP kinases), are 4-kinases and are thought to catalyse a minor route of synthesis of the multifunctional phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) from the inositide PI(5)P [2]. Here, we demonstrate the partial purification of a protein kinase that phosphorylates the type IIα PIP kinase at a single site unique to that isoform – Ser304. This kinase was identified as protein kinase CK2 (formerly casein kinase 2). Mutation of Ser304 to aspartate to mimic its phosphorylation had no effect on PIP kinase activity, but promoted both redistribution of the green fluorescent protein (GFP)-tagged enzyme in HeLa cells from the cytosol to the plasma membrane, and membrane ruffling. This effect was mimicked by mutation of Ser304 to alanine, although not to threonine, suggesting a mechanism involving the unmasking of a latent membrane localisation sequence in response to phosphorylation.     

Membrane depolarization increases membrane PtdIns(4,5)P2 levels through mechanisms involving PKC βII and PI4 kinase

In a previous study, we showed that membrane depolarization induced elevation of membrane phosphatidylinositol 4,5-bisphosphates (PtdIns(4,5)P(2), also known as PIP(2)) and subsequently increased the KCNQ2/Q3 currents expressed in Xenopus oocytes through increased PI4 kinase activity. In this study, the underlying mechanism for this depolarization-induced enhancement of PIP(2) synthesis was further investigated. Our results indicate that activation of protein kinase C (PKC) isozyme βII was responsible for the enhanced PIP(2) synthesis. We found that phorbol-12-myristate, 13-acetate (PMA), an activator of PKC, mimicked the effects of the membrane depolarization by increasing KCNQ2/Q3 activity, elevating membrane PIP(2) levels and increasing activity of PI4 kinase β. Furthermore, membrane depolarization enhanced PKC activity. The effects of both depolarization and PMA were blocked by a PKC inhibitor or PI4 kinase β RNA interference. Further results demonstrate that the depolarization selectively activated the PKC βII isoform and enhanced its interaction with PI4 kinase β. These results reveal that the depolarization-induced elevation of membrane PIP(2) is through activation of PKC and the subsequent increased activity of PI4 kinase β.