Harry B. Paddon

Membrane-bound CTP: Phosphocholine cytidylyltransferase regulates the rate of phosphatidylcholine synthesis in HeLa cells treated with unsaturated fatty acids

The influence of fatty acids on phosphatidylcholine biosynthesis in HeLa cell cultures was investigated. Oleate and other unsaturated fatty acids stimulated the incorporation of phospho[Me-3H]choline into phosphatidylcholine from 5 to 20-fold, while saturated fatty acids were without effect. Stimulation of the reaction catalyzed by CTP:phosphocholine cytidylyltransferase (CTP:cholinephosphate cytidylyltransferase, EC 2.7.7.15) by 1 mM oleate was evident within 15 min and could be reversed within 40 min after removal of the oleate-supplemented cell medium. Cytidylyltransferase activity was 11-fold higher in homogenates from cells exposed to oleate. Treatment of the HeLa cells with oleate produced almost complete translocation of the cytidylyltransferase from the cytosol to the microsomal fraction. Additional support for conversion of the cytidylyltransferase to a membrane-bound form in oleate-treated cells was obtained in studies with digitonin. Exposure of control cells to digitonin for 2 min caused the release of 60% of the total cytidylyltransferase into the medium, while oleate-treated cells leaked only 5% of the enzyme in the presence of digitonin. Finally, oleate (50 microM) was shown to promote complete aggregation of cytosolic cytidylyltransferase with microsomes from HeLa cells and 22-fold stimulation of the enzyme activity. It appears that the rate of phosphatidylcholine biosynthesis is governed by the amount of cytidylyltransferase bound to endoplasmic reticulum in HeLa cells.

B23/Nucleophosmin Serine 4 Phosphorylation Mediates Mitotic Functions of Polo-like Kinase 1

Phosphoprotein profiling by Kinetworks™ analysis of M-phase-arrested HeLa cells by nocodazole treatment revealed that a novel mitosis-specific phosphorylation event occurred in the nucleolar protein B23/nucleophosmin at a conserved Ser-4 residue. Consistent with the resemblance of the Ser-4 phosphorylation site to the Polo-like kinase 1 (Plk1) consensus recognition sequence, inhibition of Plk1 by a kinase-defective mutation (K82M) abrogated B23 Ser-4 phosphorylation, whereas activation of Plk1 by a constitutively active mutation (T210D) enhanced its phosphorylation following in vivo transfection and in vitro phosphorylation assays. Depletion of endogenous Plk1 by RNA interference abolished B23 Ser-4 phosphorylation. The physical interaction of Plk1 and B23 was further demonstrated by their co-immunoprecipitation and glutathione S-transferase fusion protein pull-down assays. Interference of Ser-4 phosphorylation of B23 induced multiple mitotic defects in HeLa cells, including aberrant numbers of centrosomes, elongation and fragmentation of nuclei, and incomplete cytokinesis. The phenotypes of B23 mutants are reminiscent of a subset of those described previously in Plk1 mutants. Our findings provide insights into the biochemical mechanism underlying the role of Plk1 in mitosis regulation through the identification of Ser-4 in B23 as a major physiological substrate of Plk1.

Identification of p42 mitogen-activated protein kinase as a tyrosine kinase substrate activated by maximal electroconvulsive shock in hippocampus

Abstract: Recent studies have demonstrated that administration of an electroconvulsive shock produces a rapid and transient increase in tyrosyl phosphorylation of a ∼40‐kDa protein in rat brain. Initial characterization of this proteins chromatographic properties indicated that it might be a member of a recently identified family of kinases, referred to as mitogen‐activated protein (MAP) kinases, that are activated by tyrosyl phosphorylation. In the present study, we have used MAP kinase antisera to assess the identity of this protein. We have found that the ∼40‐kDa phosphotyrosine‐containing protein comigrates with p42 MAP kinase (p42mapk) and not with two other 44‐kDa MAP kinase family members detected by these antisera. Western blots of proteins immunoprecipitated with MAP kinase antibodies confirm that p42mapk displays increased tyrosyl phosphorylation after an electroconvulsive stimulus. Chromatographic separation of hippocampal extracts indicates that MAP kinase activity elutes in parallel with p42mapk. Accordingly, these studies identify p42mapk as a tyrosyl kinase substrate that is activated by this stimulus and suggest that this form of MAP kinase may be selectively regulated by neuronal stimulation.

Tetradecanoyl-phorbol acetate stimulates phosphatidylcholine biosynthesis in hela cells by an increase in the rate of the reaction catalyzed by CTP: Phosphocholine cytidylyltransferase

Studies have been performed on the mechanism by which 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulates phosphatidylcholine biosynthesis in HeLa cells. The phorbol acetate did not alter the transport of choline into the cells, nor did it stimulate choline phosphorylation. When [methyl-3H]choline was added to HeLa cells for a 1 h pulse, virtually all of the radioactivity in the aqueous phase of the cell extracts became associated with phosphocholine. The addition of the phorbol ester caused an accelerated disappearance of radioactivity from phosphocholine and a concomitant increase in the incorporation into phosphatidylcholine. The radioactivity associated with CDPcholine remained low and constant throughout the experiment. These results provide strong evidence that TPA accelerates phosphatidylcholine biosynthesis in HeLa cells by stimulation of the reaction catalyzed by CTP:phosphocholine cytidylyltransferase.

Phorbol esters stimulate phosphatidylcholine biosynthesis by translocation of CTP: Phosphocholine cytidylyltransferase from cytosol to microsomes

Previous studies have demonstrated that 12-O-tetradecanoyl phorbol 13-acetate (TPA) stimulates phosphatidylcholine biosynthesis in HeLa cells. The stimulation was apparently caused by an acceleration of the reaction catalyzed by CTP:phosphocholine cytidylyltransferase (CTP:cholinephosphate cytidylyltransferase, EC 2.7.7.15) (Paddon, H.B. and Vance, D.E. (1980) Biochim. Biophys. Acta 620, 636-640). We now provide evidence that the enzyme activation is due to a translocation of the cytidylyltransferase from the cytosol to the microsomes. The rate of phospho[Me-3H]choline conversion into phosphatidylcholine was approx. 3-fold faster in HeLa cells treated with 100 nM TPA. This stimulation correlated with a 2.3-fold activation (P less than 0.05) of cytidylyltransferase in homogenates from treated cells. There was a 1.7-fold increase in the enzyme associated with microsomes (P less than 0.05) and a corresponding decrease in enzyme recovered from cytosol (P less than 0.01). The total amount of enzyme recovered from the homogenates was unchanged. Further evidence for TPA causing an increased association of cytidylyltransferase with cellular membranes was obtained when cells were treated with digitonin. The release of cytidylyltransferase into the medium was inhibited by 4-fold from cells previously treated with TPA. Similar results on phospho[Me-3H]choline incorporation into phosphatidylcholine were found with cells incubated with phorbol-12,13-dibutyrate, a water-soluble tumor-promoting agent.

A role for amplified protein kinase C activity in the pathogenesis of amyotrophic lateral sclerosis

Abstract: Amyotrophic lateral sclerosis (ALS) is a human neurodegenerative disorder of unknown origin that is characterized by progressive degeneration of corticospinal tracts and anterior horn cells in the brainstem and spinal cord. Previous studies have indicated that motoneuron degeneration associated with ALS may be triggered by mechanisms leading to increased intracellular Ca2+. In the present report, Ca2+‐activated phospholipid‐dependent protein kinase C (PKC) was evaluated in cervical spinal cords from ALS patients and control subjects. In patients who died with ALS, PKC histone H1 phosphotransferase activity was significantly increased by 330% in cytosolic‐ and 118% in particulate‐derived extracts compared with controls. This increase in PKC phosphotransferase activity appeared to be partially due to an increase in the amount of PKC protein present in ALS spinal cord tissue. PKC histone H1 phosphotransferase activities of cytosolic‐ and particulate‐derived extracts from motor and visual cortex of ALS patients and controls were not statistically different, nor were there differences in PKC histone H1 phosphotransferase activity in platelets and leukocytes. The specific nature of PKC alterations in affected regions of the CNS supports a role for PKC in the events leading to motoneuron death in sporadic ALS.

Stimulation of hepatic phosphatidylcholine biosynthesis in rats fed a high cholesterol and cholate diet correlates with translocation of CTP:Phosphocholine cytidylyltransferase from cytosol to microsomes

A new model system for the study of phosphatidylcholine biosynthesis is presented. Young rats were fed a diet that contained 5% cholesterol and 2% cholate. After 6 days there was a 2-fold increase in the concentration of plasma phospholipid (243 mg/dl compared to 132 mg/dl for control animals) and a 3-fold increase in the concentration of plasma phosphatidylcholine. The rate of phosphatidylcholine biosynthesis was measured after injection of [Me-3H]choline into the portal veins. The incorporation of tritium into choline, phosphocholine and betaine by liver was similar for experimental and control animals, whereas there was a 3-fold increased incorporation into phosphatidylcholine of the cholesterol/cholate-fed rats. The activities of the enzymes of phosphatidylcholine biosynthesis in cytosol and microsomes were assayed. The only change detected was in the cytosolic and microsomal activities of CTP: phosphocholine cytidylyltransferase which were increased more than 2-fold in specific activity. When total cytidylyltransferase activity per liver was determined, a dramatic translocation of the enzyme to microsomes was observed. The control livers had 24% of the cytidylyltransferase activity associated with microsomes, whereas this value was 61% in the livers from cholesterol/cholate-fed rats. When the cytosolic cytidylyltransferase was assayed in the presence of phospholipid, the enzyme was stimulated several-fold and the difference in specific activity between control and cholesterol/cholate-fed rats was abolished. The increased activity in cytosol appears to be the result of a 2-fold increase in the amount of phospholipid in the cytosol from cholesterol/cholate-fed rats. The data strongly support the hypothesis that the special diet stimulates phosphatidylcholine biosynthesis by causing a translocation of the cytidylyltransferase from cytosol to microsomes where it is activated.

Protein kinase C activation by platelet-activating factor is independent of enzyme translocation.

The subcellular distribution and activation state of protein kinase C (PKC) was studied after short-term exposure of rabbit platelets to platelet-activating factor (PAF). Cytosolic and nonidet P-40-solubilized particulate extracts prepared from treated platelets were subjected to analytical column chromatography on MonoQ, hydroxylapatite and Superose 6/12. PKC activity was assayed by the ability of the enzyme to phosphorylate the following substrates: (i) histone H1 in the presence of the activators calcium, diacylglycerol and phosphatidylserine; (ii) histone H1 following proteolytic activation of PKC with 0.5 micrograms trypsin/ml; and (iii) protamine in the absence of calcium and lipid. PAF treatment for 1-20 min elicited a rapid 2-4-fold activation of both cytosolic and particulate-derived PKC as assessed by all three methods. On the other hand, there were no significant PAF-induced changes in the level of [3H]phorbol-12,13-dibutyrate binding by soluble and particulate-associated PKC. Hydroxyapatite column chromatography revealed that in non-treated rabbit platelets the type II (beta) form of PKC predominated, but PAF appeared to induce a shift in the elution profile from this resin. The stability of the PAF activation of PKC to column chromatography and the altered binding affinity to hydroxylapatite indicated that the stimulation might be a consequence of covalent modification, albeit minor, since PKC still eluted as an 80 kDa protein from Superose 6/12. As the PAF-induced increases in the kinase activity of PKC were preserved even after proteolytic activation with trypsin, but were without effect on the phorbol ester binding activity, such a putative modification may have occurred within or near the catalytic domain of PKC. These findings imply that PAF may directly modulate the activity of preexisting membrane-associated PKC by a novel mechanism, rather than by eliciting its recruitment from the cytoplasm.

Diethylstilbestrol treatment modulates the enzymatic activities of phosphatidylcholine biosynthesis in rooster liver.

The effect of diethylstilbestrol injection on the activities of phosphatidylcholine biosynthetic enzymes in rooster liver has been determined. Choline kinase activity was stimulated within 4 h after the first hormone injection. By the third day enzyme activity reached 5.47 nmol . min-1 . mg-1 protein compared to control values (1.83 nmol . min-1 . mg-1 protein) which were unchanged during the the experiment. CTP : phosphocholine cytidylyltransferase activity was unaffected until Day 3 when its activity was 50% that of control values. When assayed in the presence of exogenous phospholipid, no significant change was noted in cytidylyltransferase activity. The activity of CDPcholine : 1,2-diacylglycerol phosphocholinetransferase was not altered by the hormone injections. The activity of phosphatidylethanolamine-N-methyltransferase gradually increased so that by Day 3, the enzyme activity was elevated 2-fold (0.12 to 0.24 nmol methyl group transferred per mg microsomal protein). These results are consistent with earlier in vivo studies (Vigo, C. and Vance, D.E. (1981) Eur. J. Biochem., in the press) that indicated a stimulation of phosphatidylcholine biosynthesis via CDPcholine during the first 2 days of diethylstilbestrol injection and inhibition on the third day.

Histamine releases PGI2 from human pulmonary artery

Histamine caused a triphasic response of human pulmonary artery strips in vitro, consisting of a small initial contraction followed by pronounced relaxation preceding a second contractile response. These characteristics were not seen with other contractile stimuli including 5-hydroxytryptamine, leukotriene D4, and KCl. The relaxant component of this response was ablated by removal of endothelium from the vascular strips or by pretreatment of the tissue with 1 microM indomethacin. Measurement of the PGI2 degradation product 6-keto-PGF1 alpha in supernatants from histamine-challenged tissues confirmed the synthesis of PGI2. Supernatants from unstimulated or leukotriene-challenged tissues contained no detectable amounts of 6-keto-PGF1 alpha. The histamine H1 antagonist diphenydramine inhibited both the contractile and relaxant responses to histamine whereas the H2 antagonist cimetidine affected neither component. The released PGI2 significantly altered the dose-response curve to histamine without inhibiting the maximal contractile responses. We conclude that histamine induces PGI2 formation from pulmonary arterial endothelium via an H1 receptor.