Gerry T. Snoek

Phospholipid requirement of the membrane-bound Mg2+-dependent adenosinetriphosphatase in Acholeplasma laidlawii

Abstract 1. 1. Treatment of membranes of Acholeplasma laidlawii B with phospholipase A2 from pig pancreas and phospholipase C from Bacillus cereus results in complete hydrolysis of phosphatidylglycerol. 2. 2. Phosphatidlglycerol is not required for the activity of two membrane-bound enzymes: NADH oxidase and p- nitrophenylphosphatase . A slight increase in activity of those enzymes is observed upon complete hydrolysis of phosphatidlglycerol. 3. 3. 90% of the phosphatidlglycerol can be hydrolysed with phospholipases A2 and C without loss of activity of Mg2+-dependent adenosinetriphosphatase (Mg2+-ATPase). Hydrolysis of the residual 10% phosphatidlglycerol strongly reduces the Mg2+-ATPase activity. 4. 4. Modification of phosphatidlglycerol to phosphatidic acid by phospholipase D from cabbage does not effect the Mg2+-ATPase activity. 5. 5. The inactivated Mg2+-ATPase in phosphatidlglycerol-depleted membranes can be reactivated by adding phosphatidlglycerol, phosphatidic acid or phosphatidylserine but not with phosphatidylcholine, phosphatidylethanolamine nor any of the A. laidlawii lipids except phosphatidlglycerol. 6. 6. The ability to restore full Mg2+-ATPase activity in membranes which contain less than 2% of their original amount of phosphatidlglycerol is lost gradually upon prolonged incubation times. This irreversible loss of Mg2+-ATPase activity is not accompanied by a measurable hydrolysis of the residual phosphatidlglycerol. 7. 7. Reconstitution experiments show that the fatty acid composition of both the (residual) phosphatidlglycerol present in the membrane as well as the added phosphatidlglycerol, determine the activation energy of the Mg2+-ATPase and the temperature at which a break in the Arrhenius plot occurs.

Overexpression of phosphatidylinositol transfer protein alpha in NIH3T3 cells activates a phospholipase A.

In order to investigate the cellular function of the mammalian phosphatidylinositol transfer protein α (PI-TPα), NIH3T3 fibroblast cells were transfected with the cDNA encoding mouse PI-TPα. Two stable cell lines, i.e. SPI6 and SPI8, were isolated, which showed a 2- and 3-fold increase, respectively, in the level of PI-TPα. Overexpression of PI-TPα resulted in a decrease in the duration of the cell cycle from 21 h for the wild type (nontransfected) NIH3T3 (wtNIH3T3) cells and mock-transfected cells to 13–14 h for SPI6 and SPI8 cells. Analysis of exponentially growing cultures by fluorescence-activated cell sorting showed that a shorter G1 phase is mainly responsible for this decrease. The saturation density of the cells increased from 0.20 × 105 cells/cm2 for wtNIH3T3 cells to 0.53 × 105 cells/cm2 for SPI6 and SPI8 cells. However, anchorage-dependent growth was maintained as shown by the inability of the cells to grow in soft agar. Upon equilibrium labeling of the cells withmyo-[3H] inositol, the relative incorporation of radioactivity in the total inositol phosphate fraction was 2–3-fold increased in SPI6 and SPI8 cells when compared with wtNIH3T3 cells. A detailed analysis of the inositol metabolites showed increased levels of glycerophosphoinositol, Ins(1)P, Ins(2)P, and lysophosphatidylinositol (lyso-PtdIns) in SPI8 cells, whereas the levels of phosphatidylinositol (PtdIns) and phosphatidylinositol 4,5-bisphosphate were the same as those in control cells. The addition of PI-TPα to a total lysate ofmyo-[3H]inositol-labeled wtNIH3T3 cells stimulated the formation of lyso-PtdIns. The addition of Ca2+ further increased this formation. Based on these observations, we propose that PI-TPα is involved in the production of lyso-PtdIns by activating a phospholipase A acting on PtdIns. The increased level of lyso-PtdIns that is produced in this reaction could be responsible for the increased growth rate and the partial loss of contact inhibition in SPI8 and SPI6 cells. The addition of growth factors (platelet-derived growth factor, bombesin) to these overexpressers did not activate the phospholipase C-dependent degradation of phosphatidylinositol 4,5-bisphosphate.

Rapid replenishment of sphingomyelin in the plasma membrane upon degradation by sphingomyelinase in NIH3T3 cells overexpressing the phosphatidylinositol transfer protein beta.

In order to study the in vivo function of the phosphatidylinositol transfer protein beta (PI-TPbeta), mouse NIH3T3 fibroblasts were transfected with cDNA encoding mouse PI-TPbeta. Two stable cell lines were isolated (SPIbeta2 and SPIbeta8) in which the levels of PI-TPbeta were increased 16- and 11-fold respectively. The doubling time of the SPIbeta cells was about 1.7 times that of the wild-type (wt) cells. Because PI-TPbeta expresses transfer activity towards sphingomyelin (SM) in vitro, the SM metabolism of the overexpressors was investigated. By measuring the incorporation of [methyl-(3)H]choline chloride in SM and phosphatidylcholine (PtdCho), it was shown that the rate of de novo SM and PtdCho synthesis was similar in transfected and wt cells. We also determined the ability of the cells to resynthesize SM from ceramide produced in the plasma membrane by the action of bacterial sphingomyelinase (bSMase). In these experiments the cells were labelled to equilibrium (60 h) with [(3)H]choline. At relatively low bSMase concentrations (50 munits/ml), 50% of [(3)H]SM in wt NIH3T3 cells was degraded, whereas the levels of [(3)H]SM in SPIbeta cells appeared to be unaffected. Since the release of [(3)H]choline phosphate into the medium was comparable for both wt NIH3T3 and SPIbeta cells, these results strongly suggest that breakdown of SM in SPIbeta cells was masked by rapid resynthesis of SM from the ceramide formed. By increasing the bSMase concentrations to 200 munits/ml, a 50% decrease in the level of [(3)H]SM in SPIbeta cells was attained. During a recovery period of 6 h (in the absence of bSMase) the resynthesis of SM was found to be much more pronounced in these SPIbeta cells than in 50% [(3)H]SM-depleted wt NIH3T3 cells. After 6 h of recovery about 50% of the resynthesized SM in the SPIbeta cells was available for a second hydrolysis by bSMase. When monensin was present during the recovery period, the resynthesis of SM in bSMase-treated SPIbeta cells was not affected. However, under these conditions 100% of the resynthesized SM was available for hydrolysis. On the basis of these results we propose that, under conditions where ceramide is formed in the plasma membrane, PI-TPbeta plays an important role in restoring the steady-state levels of SM.

Phorbol ester binding and protein kinase C activity in normal and transformed human keratinocytes

Normal keratinocytes, SV40-transformed keratinocytes (SVK14), and various squamous carcinoma cell (SCC) lines have been used as an in vitro model system to study the properties of phorbol ester receptor and protein kinase C expression during keratinocyte differentiation. The cell lines used exhibit a decreasing capacity to differentiate in the order of keratinocytes approximately SVK14 greater than SCC-12F2 greater than SCC-15 greater than SCC-4; moreover, all cell lines respond to a low external Ca2+ concentration by a decreased capacity to differentiate. Normal keratinocytes exhibited the highest number of phorbol ester receptors as compared to the other cell lines, while each individual cell line exhibited a higher number of phorbol ester receptors during growth under normal Ca2+ conditions as compared to cells grown under low Ca2+ conditions. The apparent dissociation constant (Kd) demonstrated only small variations in the various cell lines. In contrast, the cytoplasmic protein kinase C activity, was found to be higher in cells grown under low Ca2+ conditions than in cells grown under normal Ca2+ conditions, indicating the absence of a causal relationship between cytoplasmic protein kinase C activity and phorbol ester receptor expression. Therefore the properties of protein kinase C have been determined in more detail in normal keratinocytes and SCC-15 cells. These studies revealed differences between protein kinase C properties from the two cell lines grown under normal and low Ca2+ conditions. The differences included the effect of phorbol 12-myristate 13-acetate (PMA) on the redistribution pattern of protein kinase C between the cytoplasmic and particulate fractions as well as the activating effect of diolein in vitro on protein kinase C activity, partly purified from particulate or cytoplasmic fractions. These observations demonstrate that the functional protein kinase C activity of keratinocytes is determined by various endogenous and exogenous activators and that these activators are modulated differently in various cell lines, under various growth conditions (low Ca2+ versus normal Ca2+).

Characterization of mouse phosphatidylinositol transfer protein expressed in Escherichia coli

The cDNA encoding mouse phosphatidylinositol transfer protein (PI-TP) was isolated by means of reverse transcriptase polymerase chain reaction. The nucleotide sequence of this cDNA has a high similarity (98%) with that of rat PI-TP; the predicted amino acid sequence is 99.6% identical to that of rat PI-TP. The cDNA encoding mouse PI-TP was cloned into the expression vector pET3d and the Escherichia coli strain BL21(DE3) was transformed with the resulting plasmid. After induction of the bacteria with isopropyl-beta-D-thiogalactopyranoside, PI-TP was efficiently expressed in the E. coli strain. It was estimated that 5% of the total soluble cell protein consisted of PI-TP. The recombinant mouse PI-TP was purified from the bacterial lysate in four steps: ammonium sulphate precipitation, anion-exchange chromatography, heparin-Sepharose affinity and gel filtration chromatography. Fractionation on the heparin-Sepharose affinity column yielded two forms: PI-TP Hepa1 and Hepa2. These two proteins have the same molecular mass of 35 kDa, both contain a phosphatidylglycerol molecule and both are recognized by anti-PI-TP antibody. Both recombinant proteins have an isoelectric point of 5.4 as compared to 5.5 for bovine brain PI-TP. Sequence analysis of the first 25 N-terminal amino acid residues showed that both forms are identical, except that PI-TP Hepa1 contains the initiator methionine which is lacking from PI-TP Hepa2. The two PI-TP forms have similar phospholipid-binding and transfer activity, comparable to that of bovine brain PI-TP. Both forms and bovine brain PI-TP are phosphorylated equally well in a Ca2+/phospholipid-dependent way by protein kinase C.

Protein kinase C and phorbol ester receptor expression related to growth and differentiation of nullipotent and pluripotent embryonal carcinoma cells

We have characterized effects of phorbol, 12-myristate 13 acetate (PMA) on growth and differentiation in a nullipotent embryonal carcinoma (EC) cell line, F9, in a pluripotent EC line, P19, and in the differentiated derivatives of these cells, In P19EC and F9EC PMA addition resulted in inhibition of growth, while in the differentiated derivates PMA was mitogenic. PMA did not induce differentiation in EC cells but potentiated the retinoic acid (RA) induced differentiation in P19EC, although, not in F9EC. Rapid morphological changes by PMA were seen in P19EC and two differentiated derivatives which represent different stages of differentiation. In F9 no rapid morphological changes were induced by PMA. Using [3H]phorbol dibutyrate as a ligand we showed that during differentiation into endoderm-like cells the number of phorbol ester receptors increases, while in epithelial-like derivatives no increase is found. In differentiated cells with an increased number of phorbol ester receptors, the cytoplasmic Ca2+- and phospholipid-dependent protein kinase (the putative receptor for phorbol esters) activity was also increased. Only in those derivatives where the number of phorbol ester receptors is increased, is the binding of epidermal growth factor (EGF) inhibited by PMA. These results suggest a relationship between levels of expression of phorbol ester receptors, cytoplasmic protein kinase C and biological effects, namely rapid morphological changes, altered growth, potentiation of RA induced differentiation, and inhibition of EGF binding.

The structure of phosphatidylinositol transfer protein α reveals sites for phospholipid binding and membrane association with major implications for its function

Elucidation of the three‐dimensional structure of phosphatidylinositol transfer protein α (PI‐TPα) void of phospholipid revealed a site of membrane association connected to a channel for phospholipid binding. Near the top of the channel specific binding sites for the phosphorylcholine and phosphorylinositol head groups were identified. The structure of this open form suggests a mechanism by which PI‐TPα preferentially binds PI from a membrane interface. Modeling predicts that upon association of PI‐TPα with the membrane the inositol moiety of bound PI is accessible from the medium. Upon release from the membrane PI‐TPα adopts a closed structure with the phospholipid bound fully encapsulated. This structure provides new insights as to how PI‐TPα may play a role in PI metabolism.

The interaction of protein kinase C and other specific cytoplasmic proteins with phospholipid bilayers

The role of lipid composition in the interaction of purified protein kinase C with large unilamellar vesicles was determined by the extent of photolabelling of the enzyme with 5-[125I]iodonaphthalene-I-azide. The protein kinase C was only slightly labelled when exposed to phosphatidylcholine (PC) liposomes. The addition of phorbol 12-myristate 13-acetate (PMA) or of diacylglycerol to the PC liposomes enhanced significantly the labelling of the protein kinase C at low calcium concentrations. A further enhancement in the photolabelling of the protein kinase C was observed in liposomes containing 2% phosphatidylserine (PS). At low calcium concentrations, the binding of the enzyme to these liposomes increased in the presence of added PMA or diacylglycerol. Raising the levels of PS beyond 2% in the liposomes did not enhance the binding of the protein kinase C. However, when the enzymatic activity of the protein kinase C was measured using basic histones as substrates, maximum phosphorylation was obtained in liposomes with a PC to PS ratio of 1. The fact that the translocation of the protein kinase C from solution to the surface of the liposomes could be monitored by its labelling with 5-iodonaphthalene 1-azide prompted us to determine whether other cytoplasmic proteins might share this property. The interaction of cytoplasmic proteins from HeLa cells with PC liposomes gave trace labelling irrespective of whether calcium was added. When the HeLa cell cytoplasmic proteins were allowed to interact with liposomes containing PS, selective 5-iodonaphthalene-1-azide photolabelling was observed in distinct proteins. Addition of calcium and of PMA or diacylglycerol modified the labelling of some but not all of these proteins. These results suggest that the methodology developed might serve to identify proteins that move to the membrane during stimulation of cells by phorbol esters or by growth factors which induce the generation of diacylglycerol. These results also suggest a role for the phospholipid composition of the plasma membrane (or any intracellular membrane) in the modulation of the activation processes of specific phospholipid-dependent proteins, in particular protein kinase C.

A phosphatidylinositol transfer protein α‐dependent survival factor protects cultured primary neurons against serum deprivation‐induced cell death

Selective neuronal loss is a prominent feature in both acute and chronic neurological disorders. Recently, a link between neurodegeneration and a deficiency in the lipid transport protein phosphatidylinositol transfer protein α (PI‐TPα) has been demonstrated. In this context it may be of importance that fibroblasts overexpressing PI‐TPα are known to produce and secrete bioactive survival factors that protect fibroblasts against UV‐induced apoptosis. In the present study it was investigated whether the conditioned medium of cells overexpressing PI‐TPα (CMα) has neuroprotective effects on primary neurons in culture. We show that CMα is capable of protecting primary, spinal cord‐derived motor neurons from serum deprivation‐induced cell death. Since the conditioned medium of wild‐type cells was much less effective, we infer that the neuroprotective effect of CMα is linked (in part) to the PI‐TPα‐dependent production of arachidonic acid metabolites. The neuroprotective activity of CMα is partly inhibited by suramin, a broad‐spectrum antagonist of G‐protein coupled receptors. Western blot analysis shows that brain cortex and spinal cord express relatively high levels of PI‐TPα, suggesting that the survival factor may be produced in neuronal tissue. We propose that the bioactive survival factor is implicated in neuronal survival. If so, PI‐TPα could be a promising target to be evaluated in studies on the prevention and treatment of neurological disorders.

Phosphatidylinositol transfer proteins: Emerging roles in cell proliferation, cell death and survival

The actual cellular functions of the highly homologous small isoforms of the phosphatidylinositol transfer proteins, PI‐TPα and PI‐TPβ have been studied using many different experimental conditions varying from in vitro experiments with purified proteins and lipid vesicles to investigations in animals. In this review, the very diverse data of these investigations have been collected and joined to propose a model for the cellular functions of PI‐TPα and PI‐TPβ. The model is based on the suggested roles of PI‐TPα and PI‐TPβ in various lipid‐mediated cellular signaling pathways and leads to the conclusion that both proteins have a regulating function in pathways involved in the proliferation, apoptosis as well as survival of cells. IUBMB Life, 56: 467‐475, 2004