Regulation of platelet activating factor synthesis: modulation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase by phosphorylation and dephosphorylation in rat spleen microsomes

1-Alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase plays an important regulatory role in the biosynthesis of platelet activating factor, a potent bioactive mediator. We tested the hypothesis that the activity of acetyltransferase may be modulated by enzymatic phosphorylation and dephosphorylation. The results showed that acetyltransferase activity in rat spleens was 2- to 3-fold higher in microsomes isolated in the presence of F-than in those isolated in the presence of Cl-. The microsomal acetyltransferase could be activated by preincubation of microsomes, isolated in the presence of Cl-, with ATP, Mg2+, and the soluble fraction from rat spleen. Addition of phosphatidylserine, diacylglycerols, plus Ca2+ further enhanced the activity. The increase in the activity of acetyltransferase was abolished by treatment of the activated microsomes with alkaline phosphatase. Conversely, the activity of acetyltransferase can be reactivated in the alkaline phosphatase-treated microsomes with incubation conditions that favor phosphorylation. Therefore, our findings suggest that acetyltransferase activity is regulated by reversible activation/inactivation through phosphorylation/dephosphorylation.     

Phosphorylation of 69-kDa choline acetyltransferase at threonine 456 in response to amyloid-beta peptide 1-42

Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons. In the brain, these neurons are especially vulnerable to effects of beta-amyloid (A beta) peptides. Choline acetyltransferase is a substrate for several protein kinases. In the present study, we demonstrate that short term exposure of IMR32 neuroblastoma cells expressing human choline acetyltransferase to A beta-(1-42) changes phosphorylation of the enzyme, resulting in increased activity and alterations in its interaction with other cellular proteins. Using mass spectrometry, we identified threonine 456 as a new phosphorylation site in choline acetyltransferase from A beta-(1-42)-treated cells and in purified recombinant ChAT phosphorylated in vitro by calcium/calmodulin-dependent protein kinase II (CaM kinase II). Whereas phosphorylation of choline acetyltransferase by protein kinase C alone caused a 2-fold increase in enzyme activity, phosphorylation by CaM kinase II alone did not alter enzyme activity. A 3-fold increase in choline acetyltransferase activity was found with coordinate phosphorylation of threonine 456 by CaM kinase II and phosphorylation of serine 440 by protein kinase C. This phosphorylation combination was observed in choline acetyltransferase from A beta-(1-42)-treated cells. Treatment of cells with A beta-(1-42) resulted in two phases of activation of choline acetyltransferase, the first within 30 min and associated with phosphorylation by protein kinase C and the second by 10 h and associated with phosphorylation by both CaM kinase II and protein kinase C. We also show that choline acetyltransferase from A beta-(1-42)-treated cells co-immunoprecipitates with valosin-containing protein, and mutation of threonine 456 to alanine abolished the A beta-(1-42)-induced effects. These studies demonstrate that A beta-(1-42) can acutely regulate the function of choline acetyltransferase, thus potentially altering cholinergic neurotransmission.     

Phosphorylation of Rat Brain Choline Acetyltransferase and Its Relationship to Enzyme Activity

Abstract— Choline acetyltransferase catalyzes the formation of acetylcholine from choline and acetyl-CoA in cholin-ergic neurons. The present study examined conditions for modulation of kinase-mediated phosphorylation of this enzyme. By using a monospecific polyclonal rabbit anti-human choline acetyltransferase antibody to immunoprecipi-tate cytosolic and membrane-associated subcellular pools of enzyme from rat hippocampal synaptosomes, we determined that only the cytosolic fraction of the enzyme (67,000 ± 730 daltons) was phosphorylated under basal, unstimulated conditions. The quantity of this endogenous phosphoprotein was dependent, in part, upon the level of intracellular calcium, with ³²P_i incorporation into the enzyme in nerve terminals incubated in nominally calcium-free medium only 43 ± 7% of control. The corresponding enzymatic activity of cytosolic choline acetyltransferase did not appear to be altered by lowered cytosolic calcium, whereas membrane-associated choline acetyltransferase activity was decreased to 58 ± 11 % of control. Depolarization of synaptosomes with 50 μ M veratridine neither altered the extent of phosphorylation or specific activity of cytosolic choline acetyltransferase, nor induced detectable phosphorylation of membrane-associated choline acetyltransferase, although the specific activity of the membrane-associated enzyme was increased to 132 ± 5% of control. In summary, phosphorylation of choline acetyltransferase does not appear to regulate cholinergic neurotransmission by a direct action on catalytic activity of the enzyme.     

Modulation of lyso-platelet-activating factor: acetyl-CoA acetyltransferase from rat splenic microsomes. The role of calcium ions

The enzyme lyso-platelet-activating factor: acetyl-CoA acetyltransferase (EC 2.3.1.67) was assayed in microsomal fractions from rat spleens. The addition of micromolar Ca2+ rapidly enhanced acetyltransferase activity and this activation was reversed by the addition of EGTA in excess of Ca2+. The effect of Ca2+ was on the apparent Km of the enzyme for the substrate acetyl-CoA without showing any significant effect on the Vmax of the acetylation reaction. When microsomes were isolated in the presence of 5 mM EGTA, to remove endogenous calmodulin, the same enhancing effect of Ca2+ on the acetylation reaction was observed. The addition of exogenous calmodulin to this preparation had no effect on the enzyme activity. Preincubation of spleen microsomes with the calmodulin inhibitor trifluoperazine decreased acetyltransferase in both the presence and the absence of Ca2+, indicating an effect of this drug independently of calmodulin. The addition of Mg-ATP to the assay mixture also had no effect on the acetylation reaction. These data suggest that Ca2+ modulates acetyltransferase activity from rat spleen microsomes by a mechanism that seems to be independent of calmodulin or protein phosphorylation.     

Nuclear Rho kinase, ROCK2, targets p300 acetyltransferase

Rho-associated coiled-coil protein kinase (ROCK) is an effector for the small GTPase Rho and plays a pivotal role in diverse cellular activities, including cell adhesion, cytokinesis, and gene expression, primarily through an alteration of actin cytoskeleton dynamics. Here, we show that ROCK2 is localized in the nucleus and associates with p300 acetyltransferase both in vitro and in cells. Nuclear ROCK2 is present in a large protein complex and partially cofractionates with p300 by gel filtration analysis. By immunofluorescence, ROCK2 partially colocalizes with p300 in distinct insoluble nuclear structures. ROCK2 phosphorylates p300 in vitro, and nuclear-restricted expression of constitutively active ROCK2 induces p300 phosphorylation in cells. p300 acetyltransferase activity is dependent on its phosphorylation status in cells, and p300 phosphorylation by ROCK2 results in an increase in its acetyltransferase activity in vitro. These observations suggest that nucleus-localized ROCK2 targets p300 for phosphorylation to regulate its acetyltransferase activity.     

Acetylation of polyamines in chicken brain and retina

Both spermidine and spermine are acetylated in chicken brain and retina. From spermidine, more N1-acetylspermidine than N8-acetylspermidine is formed by both the brain and the retinal cytosol. Km for spermidine is similar with the enzyme preparation of the two tissues, but that for spermine is lower with the retinal preparation. Both tissues contain an activity able to reduce spermidine acetyltransferase activity. Both alkaline phosphatase and cAMP-dependent protein kinase (catalytic subunit) are able to inactivate the spermidine acetyltransferase activity of both tissues. Spermidine acetyltransferase activity and polyamine levels have been measured in both brain and retina during embryonic life. Only in the last part of the development can enzyme activity be correlated with the retina spermidine and spermine concentration.     

Purification and properties of carnitine acetyltransferase from human liver

Carnitine acetyltransferase was purified from the supernatant obtained after centrifugation of human liver homogenate to a final specific activity of 78.75 unit.mg-1 with acetyl-CoA as a substrate. Human carnitine acetyltransferase is a monomer of 60.5 kDa with maximum activity in the presence of propionyl-CoA and a pH optimum of 8.7. Apparent Km values for acetyl-CoA are three times lower than for decanoyl-CoA. Km values for L-carnitine in the presence of acetyl-CoA are six times lower than in the presence of decanoyl-CoA. Km values for acetylcarnitine are three times lower than for octanoylcarnitine. The polyclonal antibodies against human carnitine acetyltransferase recognize a 60.5-kDa peptide in the purified preparation of human liver and brain homogenates and in immunoblots of mitochondrial and peroxisomal fractions from human liver. Immunoprecipitation and SDS/PAGE analysis of 35S-labelled proteins produced by human fibroblasts indicate that mitochondrial carnitine acetyltransferase is synthesized as a precursor of 65 kDa. We also purified carnitine acetyltransferase from the pellet obtained after centrifugation of liver homogenate. The pellet was extracted by sonication in the presence of 0.5% Tween 20. The chromatographic procedures for the purification and the kinetic, physical and immunological properties of pellet-extracted carnitine acetyltransferase are similar to those of carnitine acetyltransferase purified from the supernatant of human liver homogenate.     

Enzymes of platelet activating factor synthesis in brain

In this review, evidence is summarized for the production of PAF in brain, in response to stimulation associated with pathology. As well, there is a growing literature on the duality of actions of this lipid autocoid upon nervous tissue, indicated by extracellular and intracellular actions and binding sites for PAF in brain. The metabolic routes to PAF can be divided into the de novo and remodelling pathways of synthesis. The de novo route consists of 1-alkyl glycerophosphate acetyltransferase, and the subsequent actions of distinct phosphohydrolase and cholinephosphotransferase activities. This acetyltransferase can be activated by phosphorylation, and inhibited by MgATP and fatty acyl CoA thioesters, inhibitions which have particular relevance to brain ischemia. There is also evidence that the cholinephosphotransferase is controlled by phosphorylation, and regulated by levels of CDP-choline. The remodelling pathway to PAF relies upon the actions of phospholipase A₂ or CoA-independent transacylases to generate the l-alkyl glycerophosphorylcholine, as substrate for a distinct acetyltransferase. Following stimulation, rising intracellular calcium may trigger arachidonate selective cytosolic phospholipase activity which leads to increased PAF synthesis. The l-alkyl glycerophosphocholine acetyltransferase activity is quite small in brain in comparison with the de novo acetyltransferase activity, and is also controlled by phosphorylation. Evidence has been presented for the actions of both pathways in brain, in response to biologically relevant stimulation pertinent to the disease state.Special issue dedicated to Dr. Leon S. Wolfe.     

Acetyl-CoA:1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase is directly activated by p38 kinase

Acetyl-CoA:1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase, along with phospholipase A2, is a key regulator of platelet-activating factor biosynthesis via the remodeling pathway. We have now obtained evidence in human neutrophils indicating that this enzyme is regulated by a specific member of the mitogen-activated protein kinases, namely the p38 kinase. We earlier demonstrated that tumor necrosis factor-alpha (TNF-alpha) as well as N-formyl-methionyl-leucyl-phenylalanine treatment leads to increased phosphorylation and activation of p38 kinase in human neutrophils. Strikingly, in the present study these stimuli increased the catalytic activity of acetyltransferase up to 3-fold, whereas 4-phorbol 12-myristate 13-acetate, which activates the extracellular-regulated kinases (ERKs) but not p38 kinase, had no effect. Furthermore, a selective inhibitor of p38 kinase, SB 203580, was able to abolish the TNF-alpha- and N-formyl-methionyl-leucyl-phenylalanine-induced activation of acetyltransferase. The same effect was not observed in the presence of an inhibitor that blocked ERK activation (PD 98059). Complementing the findings in intact cells, we have shown that recombinant, activated p38 kinase added to microsomes in the presence of Mg2+ and ATP increased acetyltransferase activity to the same degree as in microsomes obtained from TNF-alpha-stimulated cells. No activation of acetyltransferase occurred upon treatment of microsomes with either recombinant, activated ERK-1 or ERK-2. Finally, the increases in acetyltransferase activity induced by TNF-alpha could be ablated by treating the microsomes with alkaline phosphatase. Thus acetyltransferase appears to be a downstream target for p38 kinase but not ERKs. These data from whole cells as well as cell-free systems fit a model wherein stimulus-induced acetyltransferase activation is mediated by a phosphorylation event catalyzed directly by p38 kinase.     

Overexpression of phospholipid hydroperoxide glutathione peroxidase modulates acetyl-CoA, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase activity

The synthesis of platelet-activating factor (PAF) by -stimulated RBL-2H3 cells was significantly suppressed by overexpression of phospholipid hydroperoxide glutathione peroxidase (PHGPx). When the cells overexpressing PHGPx (L9 cells) were pretreated with diethyl maleate, which reduces PHGPx activity, PAF synthesis upon stimulation rose to levels seen in mock-transfected cells (S1 cells). Hydroperoxide levels, which are reduced in L9 cells, are involved in regulating PAF synthesis, because the addition of hydroperoxyeicosatetraenoic acid increased PAF production in -stimulated L9 cells to control cell levels. The activity of acetyl-CoA:1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase, which is involved in the last step of PAF synthesis, is also reduced in L9 cells. p38 kinase inhibitors block acetyltransferase activity in normal -stimulated cells, suggesting that p38 kinase is involved in regulating acetyltransferase activity. Recombinant active p38 kinase activates acetyltransferase, whereas alkaline phosphatase reverses this, suggesting p38 kinase directly phosphorylates acetyltransferase. p38 kinase phosphorylation is blocked in L9 cells, indicating that high hydroperoxide levels are needed for the activation of p38 kinase. Thus, intracellular hydroperoxide levels participate in regulating p38 kinase phosphorylation, which in turn controls the activation of acetyltransferase and thus the synthesis of PAF. These observations suggest that PHGPx is an important component of the mechanisms regulating inflammation.     

Tip60 acetyltransferase activity is controlled by phosphorylation

Here we show that the phosphorylation of histone acetyltransferase Tip60, a target of human immunodeficiency virus, type 1-encoded transactivator Tat, plays a crucial role in the control of its catalytic activity. Baculovirus-based expression and purification of Tip60 combined with mass spectrometry allowed the identification of serines 86 and 90 as two major sites of phosphorylation in vivo. The phosphorylation of Tip60 was found to modulate its histone acetyltransferase activity. One of the identified phosphorylated serines, Ser-90, was within a consensus cyclin B/Cdc2 site. Ser-90 was specifically phosphorylated in vitro by the cyclin B/Cdc2 complex. Accordingly, the phosphorylation of Tip60 was enhanced after drug-induced arrest of cells in G(2)/M. This G(2)/M-dependent phosphorylation of Tip60 was abolished by treating cells with a specific inhibitor of the cyclin-dependent kinase, roscovitin. All together, these results strongly suggest a G(2)/M-dependent control of Tip60 activity.     

Ibotenic acid-induced lesions of the medial septum increase hippocampal membrane associated protein kinase c activity and reduce acetylcholine synthesis: prevention by a phosphatidylcholine/vitamin B12 diet

Ibotenic acid infusion into the medial septum (MS) results in biochemical alterations in the hippocampus. The biochemical events involved in this neuronal lesion are poorly understood. We investigated the effect of a purified diet supplemented with egg phosphatidylcholine (PC) and vitamin B(12) on ibotenic acid-medicated biochemical changes in the rat hippocampus and crude synaptosomal membranes. Male Wistar rats with this MS lesion were fed a purified diet (control diet) or a purified diet supplemented with 5.7 g PC and 125 microg vitamin B(12) per 100 g (experimental diet) for 18 days. Sham-operated rats were fed the control diet. Compared with the sham-operated rats, MS-lesioned rats fed the control diet showed increased activity of membrane-bound protein kinase C (PKC), decreased activity of choline acetyltransferase, and decreased concentrations of acetylcholine in the hippocampus. The ratio of cholesterol to phospholipid in the crude synaptic membrane was lower in the lesioned rats than in the sham-operated rats, but this was not accompanied by any alteration in membrane lipid fluidity. MS-lesioned rats fed the experimental diet showed lowered PKC activity and elevated acetylcholine concentrations than did rats fed the control diet, but there were no significant effects on choline acetyltransferase activity and the lipid ratio. The ibotenic acid-mediated elevation of PKC activity was observed as early as 2 days postinjury in the control diet-fed rats but not in the experimental diet-fed rats. We propose that ibotenic acid mediates pathophysiologic actions through the activation of PKC and that PC combined with vitamin B(12) ameliorates the second messenger-mediated injury.     

Biosynthesis of paf-acether. IX. Role for a phosphorylation-dependent activation of acetyltransferase in antigen-stimulated mouse mast cells

Mouse bone marrow-derived mast cells passively sensitized with monoclonal IgE released paf-acether (platelet-activating factor) and beta-hexosaminidase when challenged with the specific antigen. The formation and the release of paf-acether followed an early increase in the activity of the acetyltransferase, the main enzyme in paf-acether biosynthesis. The antigen-induced activation of the acetyltransferase was dependent on physiologic temperature and on the presence of Ca2+. By using microsomal fractions from unchallenged and challenged mast cells, the Vmax values were 3.5 and 12.0 nmol/min/mg of protein, respectively, whereas in both cases a Km value for acetyl-coenzyme A of 172 microM was measured. The stimulation of acetyltransferase could be mimicked in vitro under experimental conditions which favor phosphorylation, i.e. adding ATP and Mg2+ to lysates from unchallenged mast cells. In contrast, ATP and Mg2+ were uneffective on lysates from challenged cells that exhibited high level of acetyltransferase activity, suggesting that phosphorylation of the enzyme already took place at the time of cell stimulation. Moreover, addition of alkaline phosphatase to microsomal fraction obtained from either antigen-challenged mouse bone marrow-derived mast cells or unchallenged cells, resulted in 52% and 43% loss of acetyltransferase activity, respectively. Phorbol myristate acetate treatment of cells doubled the enzyme activity supporting the phosphorylation hypothesis. Thus, we report on the immunologic activation of a key enzyme for paf-acether synthesis and on the mechanism of this activation in a pure mast cell population. A link between bridging of IgE receptors and the activation of an enzyme critical to the formation of a lipid mediator is thereby evidenced.     

Functional characterization of phosphorylation of 69-kDa human choline acetyltransferase at serine 440 by protein kinase C

Choline acetyltransferase, the enzyme that synthesizes the transmitter acetylcholine in cholinergic neurons, is a substrate for protein kinase C. In the present study, we used mass spectrometry to identify serine 440 in recombinant human 69-kDa choline acetyltransferase as a protein kinase C phosphorylation site, and site-directed mutagenesis to determine that phosphorylation of this residue is involved in regulation of the enzyme's catalytic activity and binding to subcellular membranes. Incubation of HEK293 cells stably expressing wild-type 69-kDa choline acetyltransferase with the protein kinase C activator phorbol 12-myristate 13-acetate showed time- and dose-related increases in specific activity of the enzyme; in control and phorbol ester-treated cells, the enzyme was distributed predominantly in cytoplasm (about 88%) with the remainder (about 12%) bound to cellular membranes. Mutation of serine 440 to alanine resulted in localization of the enzyme entirely in cytoplasm, and this was unchanged by phorbol ester treatment. Furthermore, activation of mutant enzyme in phorbol ester-treated HEK293 cells was about 50% that observed for wild-type enzyme. Incubation of immunoaffinity purified wild-type and mutant choline acetyltransferase with protein kinase C under phosphorylating conditions led to incorporation of [(32)P]phosphate, with radiolabeling of mutant enzyme being about one-half that of wild-type, indicating that another residue is phosphorylated by protein kinase C. Acetylcholine synthesis in HEK293 cells expressing wild-type choline acetyltransferase, but not mutant enzyme, was increased by about 17% by phorbol ester treatment.     

Enhanced phosphorylation of a nucleolar 110-kDa protein in rat liver by dietary manipulation

RNA polymerase 1 activity and nucleolar volume have been reported to increase in hepatocytes from rats fed a protein-free diet. Phosphorylation in vitro of a 110-kDa protein was enhanced in nuclei and nucleoli from livers of rats fed a protein-free diet. In nuclear extracts the 110-kDa protein in heat-treated nuclei was much more phosphorylated than from control liver. In contrast, casein kinase activity in the nuclear extract from control liver was comparable to that from livers of rats fed a protein-free diet. Nuclear extracts from control rat liver and livers of rats fed a protein-free diet were fractionated by DEAE-cellulose column chromatography. Casein kinase II (NII) eluted at around 0.17 M NaCl scarcely phosphorylates the 110-kDa protein. Chromatography of the nuclear extract from livers of rats fed a protein-free diet, but not from control liver, yielded fractions which eluted at 0.21-0.25 M NaCl and predominantly phosphorylated the 110-kDa protein. The phosphorylation of 110-kDa protein was not appreciably affected by a heparin concentration of 5 micrograms/ml, which completely inhibited casein kinase II. In addition, phosphorylation of the 110-kDa protein in liver nucleoli from rats fed a protein-free diet showed a lower sensitivity to heparin than that in control rat liver nucleoli. These results suggest that enhanced phosphorylation of the nuclear 110-kDa protein in livers from rats fed a protein-free diet is due to the induction of a 110-kDa protein kinase distinct from casein kinase II.     

Modulation of lyso-platelet-activating factor:acetyl-CoA acetyltransferase from rat splenic microsomes. The role of calcium ions

The enzyme lyso-platelet-activating factor:acetyl-CoA acetyltransferase (EC 2.3.1.67) was assayed in microsomal fractions from rat spleens. The addition of micromolar Ca²⁺ rapidly enhanced acetyltransferase activity and this activation was reversed by the addition of EGTA in excess of Ca²⁺. The effect of Ca²⁺ was on the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of the enzyme for the substrate acetyl-CoA without showing any significant effect on the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the acetylation reaction. When microsomes were isolated in the presence of 5 mM EGTA, to remove endogenous calmodulin, the same enhancing effect of Ca²⁺ on the acetylation reaction was observed. The addition of exogenous calmodulin to this preparation had no effect on the enzyme activity. Preincubation of spleen microsomes with the calmodulin inhibitor trifluoperazine decreased acetyltransferase in both the presence and the absence of Ca²⁺, indicating an effect of this drug independently of calmodulin. The addition of Mg-ATP to the assay mixture also had no effect on the acetylation reaction. These data suggest that Ca²⁺ modulates acetyltransferase activity from rat spleen microsomes by a mechanism that seems to be independent of calmodulin or protein phosphorylation.     

3-Hydroxy-3-methylglutaryl-coenzyme A reductase. A comparison of the modulation in vitro by phosphorylation and dephosphorylation to modulation of enzyme activity by feeding cholesterol- or cholestyramine-supplemented diets

The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hydroxymethylglutaryl-CoA reductase) was considerably inhibited during incubation with ATP+Mg²⁺. The inactivated enzyme was reactivated on further incubation with partially purified cytosolic phosphoprotein phosphatase. The inactivation was associated with a decrease in the apparent K_m of the reductase for hydroxymethylglutaryl-CoA, and this was reversed on reactivation. The slight increase in activity observed during incubation of microsomal fraction without ATP was not associated with a change in apparent K_m and, unlike the effect of the phosphatase, was not inhibited by NaF. Liver microsomal fraction from rats given cholesterol exhibited a low activity of hydroxymethylglutaryl-CoA reductase with a low apparent K_m for hydroxymethylglutaryl-CoA. Mícrosomal fraction from rats fed cholestyramine exhibited a high activity with a high K_m. To discover whether these changes had resulted from phosphorylation and dephosphorylation of the reductase, microsomal fraction from rats fed the supplemented diets and the standard diet were inactivated with ATP and reactivated with phosphoprotein phosphatase. Inactivation reduced the maximal activity of the reductase in each microsomal preparation and also reduced the apparent K_m for hydroxymethylglutaryl-CoA. There was no difference between the preparations in the degree of inactivation produced by ATP. Treatment with phosphatase restored both the maximal activity and the apparent K_m of each preparation, but never significantly increased the activity above that observed with untreated microsomal fraction. It is concluded that hydroxymethylglutaryl-CoA reductase in microsomal fraction prepared by standard procedures is almost entirely in the dephosphorylated form, and that the difference in kinetic properties in untreated microsomal fraction from rats fed the three diets cannot be explained by differences in the degree of phosphorylation of the enzyme.     

Tyr-94 Phosphorylation Inhibits Pyruvate Dehydrogenase Phosphatase 1 and Promotes Tumor Growth

Many cancer cells rely more on aerobic glycolysis (the Warburg effect) than mitochondrial oxidative phosphorylation and catabolize glucose at a high rate. Such a metabolic switch is suggested to be due in part to functional attenuation of mitochondria in cancer cells. However, how oncogenic signals attenuate mitochondrial function and promote the switch to glycolysis remains unclear. We previously reported that tyrosine phosphorylation activates and inhibits mitochondrial pyruvate dehydrogenase kinase (PDK) and phosphatase (PDP), respectively, leading to enhanced inhibitory serine phosphorylation of pyruvate dehydrogenase (PDH) and consequently inhibition of pyruvate dehydrogenase complex (PDC) in cancer cells. In particular, Tyr-381 phosphorylation of PDP1 dissociates deacetylase SIRT3 and recruits acetyltransferase ACAT1 to PDC, resulting in increased inhibitory lysine acetylation of PDHA1 and PDP1. Here we report that phosphorylation at another tyrosine residue, Tyr-94, inhibits PDP1 by reducing the binding ability of PDP1 to lipoic acid, which is covalently attached to the L2 domain of dihydrolipoyl acetyltransferase (E2) to recruit PDP1 to PDC. We found that multiple oncogenic tyrosine kinases directly phosphorylated PDP1 at Tyr-94, and Tyr-94 phosphorylation of PDP1 was common in diverse human cancer cells and primary leukemia cells from patients. Moreover, expression of a phosphorylation-deficient PDP1 Y94F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at different tyrosine residues inhibits PDP1 through independent mechanisms, which act in concert to regulate PDC activity and promote the Warburg effect.