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1.
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.  相似文献   

2.
Activity of the mammalian pyruvate dehydrogenase complex (PDC) is regulated by phosphorylation-dephosphorylation of three serine residues (designated site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) in the alpha subunit of the pyruvate dehydrogenase (E1) component. Substitutions of the phosphorylation sites were generated by site-directed mutagenesis. Glutamate (S1E) and aspartate (S1D) substitutions at site 1 resulted in the complete loss of PDC activity; however, these mutants were variably active in the decarboxylation and 2,6-dichlorophenolindophenol assays. S1Q had only 3% of wild-type PDC activity. The apparent K(m) values for pyruvate increased for the mutants of site 1 when determined in the 2,6-dichlorophenolindophenol assay. The substitutions at sites 2 and 3 caused only moderate reductions in activity in the three assays. S3E had a 27-fold increase in the apparent K(m) for thiamine pyrophosphate and 8-fold increase in the K(i) for pyrophosphate. Site 3 was almost completely protected from phosphorylation by thiamine pyrophosphate. The results show that the size rather than negative charge of the substituted amino acid residue affects the active site of E1 and that modification of each of the three serine residues affect the active site in a site-specific manner for its ability to bind the cofactor and substrates.  相似文献   

3.
Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of the matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth.  相似文献   

4.
Pyruvate dehydrogenase E1α (PDHA1) is the first component enzyme of the pyruvate dehydrogenase (PDH) complex that transforms pyruvate, via pyruvate decarboxylation, into acetyl-CoA that is subsequently used by both the citric acid cycle and oxidative phosphorylation to generate ATP. As such, PDH links glycolysis and oxidative phosphorylation in normal as well as cancer cells. Herein we report that SIRT3 interacts with PDHA1 and directs its enzymatic activity via changes in protein acetylation. SIRT3 deacetylates PDHA1 lysine 321 (K321), and a PDHA1 mutant mimicking a deacetylated lysine (PDHA1K321R) increases PDH activity, compared to the K321 acetylation mimic (PDHA1K321Q) or wild-type PDHA1. Finally, PDHA1K321Q exhibited a more transformed in vitro cellular phenotype compared to PDHA1K321R. These results suggest that the acetylation of PDHA1 provides another layer of enzymatic regulation, in addition to phosphorylation, involving a reversible acetyllysine, suggesting that the acetylome, as well as the kinome, links glycolysis to respiration.  相似文献   

5.
Function of the nonidentical subunits of mammalian pyruvate dehydrogenase   总被引:7,自引:0,他引:7  
The pyruvate dehydrogenase (PDH) component of the bovine kidney pyruvate dehydrogenase complex (PDC) contains two nonidentical subunits. PDH catalyzes the decarboxylation of pyruvate to produce α-hydroxyethylthiamine-PP (HETPP) and the reductive acetylation of the lipoyl moieties of dihydrolipoyl transacetylase with HETPP. Phosphorylation of PDH with PDH kinase and ATP markedly inhibits the first reaction but does not inhibit the second reaction. Since the α-subunit but not the β-subunit of PDH undergoes phosphorylation, these results suggest that the α-subunit catalyzes the first reaction and the β-subunit catalyzes the second reaction. Thiamine-PP reduces the rate of phosphorylation of PDC by PDH kinase and ATP. Phosphorylation of PDC increases the KD of the PDC-Mg-thiamine-PP complex about 12-fold. It appears that the thiamine-PP binding site and the phosphorylation site on PDH influence each other and that HETPP is bound to PDH in a different orientation or possibly at a different site than is thiamine-PP.  相似文献   

6.
7.
Unravelling the molecular basis of capacitation is crucial to our understanding the basis of acquisition of fertilization competence by spermatozoa. In two recent studies, we have demonstrated that dihydrolipoamide dehydrogenase, which is a post-pyruvate metabolic enzyme and one of the components of pyruvate dehydrogenase complex, undergoes capacitation-dependent tyrosine phosphorylation, and that the activity of the enzyme correlates with capacitation events in the hamster spermatozoa. However, it is not clear as to whether other components of the pyruvate dehydrogenase complex are also crucial for sperm capacitation. In this report, we have identified pyruvate dehydrogenase A2 (PDHA2), a constituent of pyruvate dehydrogenase A (PDHA), which is a component of pyruvate dehydrogenase complex that exhibits tyrosine phosphorylation during hamster spermatozoal capacitation. This is the first report showing that hamster sperm PDHA2 is a testis-specific phosphotyrosine that is associated with the fibrous sheath of hamster spermatozoa. The localization of PDHA2 in spermatozoa was investigated using antibodies to PDHA, which is the active tetrameric protein that consists of a homodimer of PDHA2 and PDHB. Both immunofluorescence and confocal studies indicated a unique non-canonical, extramitochondrial localization for PDHA in the principal piece of hamster spermatozoa. It was also observed that PDHA colocalized with AKAP4 in the fibrous sheath of the spermatozoon. The enzymatic activity of PDHA was positively correlated with hyperactivation but not with the acrosome reaction. Given the localization of PDHA and the evidence that its activity correlates positively with hyperactivation and that its PDHA2 subunit exhibits capacitation-associated protein tyrosine phosphorylation, it appears that PDHA2 is associated with the process of capacitation.  相似文献   

8.
Activity of the mammalian pyruvate dehydrogenase complex is regulated by phosphorylation-dephosphorylation of three specific serine residues (site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) of the alpha subunit of the pyruvate dehydrogenase (E1) component. Phosphorylation is carried out by four pyruvate dehydrogenase kinase (PDK) isoenzymes. Specificity of the four mammalian PDKs toward the three phosphorylation sites of E1 was investigated using the recombinant E1 mutant proteins with only one functional phosphorylation site present. All four PDKs phosphorylated site 1 and site 2, however, with different rates in phosphate buffer (for site 1, PDK2 > PDK4 approximately PDK1 > PDK3; for site 2, PDK3 > PDK4 > PDK2 > PDK1). Site 3 was phosphorylated by PDK1 only. The maximum activation by dihydrolipoamide acetyltransferase was demonstrated by PDK3. In the free form, all PDKs phosphorylated site 1, and PDK4 had the highest activity toward site 2. The activity of the four PDKs was stimulated to a different extent by the reduction and acetylation state of the lipoyl moieties of dihydrolipoamide acetyltransferase with the maximum stimulation of PDK2. Substitution of the site 1 serine with glutamate, which mimics phosphorylation-dependent inactivation of E1, did not affect phosphorylation of site 2 by four PDKs and of site 3 by PDK1. Site specificity for phosphorylation of four PDKs with unique tissue distribution could contribute to the tissue-specific regulation of the pyruvate dehydrogenase complex in normal and pathophysiological states.  相似文献   

9.
Regulation of Stat3 activation by MEK kinase 1   总被引:6,自引:0,他引:6  
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10.
The pyruvate dehydrogenase multienzyme complex (PDC) is a key regulatory point in cellular metabolism linking glycolysis to the citric acid cycle and lipogenesis. Reversible phosphorylation of the pyruvate dehydrogenase enzyme is a critical regulatory mechanism and an important point for monitoring metabolic activity. To directly determine the regulation of the PDC by phosphorylation, we developed a complete set of phospho-antibodies against the three known phosphorylation sites on the E1 alpha subunit of pyruvate dehydrogenase (PDHE1α). We demonstrate phospho-site specificity of each antibody in a variety of cultured cells and tissue extracts. In addition, we show sensitivity of these antibodies to PDH activity using the pyruvate dehydrogenase kinase-specific inhibitor dichloroacetate. We go on to use these antibodies to assess PDH phosphorylation in a patient suffering from Leigh’s syndrome. Finally, we observe changes in individual phosphorylation states following a small molecule screen, demonstrating that these reagents should be useful for monitoring phosphorylation of PDHE1α and, therefore, overall metabolism in the disease state as well as in response to a myriad of physiological and pharmacological stimuli.  相似文献   

11.
ACK1 is a nonreceptor tyrosine kinase that associates specifically with Cdc42. Relatively few ACK1 substrates and interacting proteins have been identified. In this study, we demonstrated that ACK1 phosphorylates the Wiskott-Aldrich syndrome protein (WASP), a Cdc42 effector that plays an important role in the formation of new actin filaments. ACK1 and WASP interact in intact cells, and overexpression of ACK1 promotes WASP phosphorylation. Phosphorylation of WASP in vitro was enhanced by the addition of Cdc42 or phosphatidylinositol 4,5-biphosphate, presumably due to release of the autoinhibitory interactions in WASP. Surprisingly, when we mapped the sites of WASP phosphorylation, we found that ACK1 possesses significant serine kinase activity toward WASP (directed at Ser-242), as well as tyrosine kinase activity directed at Tyr-256. A serine peptide derived from the Ser-242 WASP phosphorylation site is also a substrate for ACK1. ACK1 expressed in bacteria retained its serine kinase activity, eliminating the possibility of contamination with a copurifying kinase. Serine phosphorylation of WASP enhanced the ability of WASP to stimulate actin polymerization in mammalian cell lysates. Thus, the tyrosine kinase ACK1 acts as a dual specificity kinase toward this substrate. In contrast to other dual specificity kinases that more closely resemble Ser/Thr kinases, ACK1 is a tyrosine kinase with an active site that can accommodate both types of hydroxyamino acids in substrates.  相似文献   

12.
Glucose-induced insulin secretion from pancreatic β-cells depends on mitochondrial activation. In the organelle, glucose-derived pyruvate is metabolised along the oxidative and anaplerotic pathway to generate downstream signals leading to insulin granule exocytosis. Entry into the oxidative pathway is catalysed by pyruvate dehydrogenase (PDH) and controlled in part by phosphorylation of the PDH E1α subunit blocking enzyme activity. We find that glucose but not other nutrient secretagogues induce PDH E1α phosphorylation in INS-1E cells and rat islets. INS-1E cells and primary β-cells express pyruvate dehydrogenase kinase (PDK) 1, 2 and 3, which mediate the observed phosphorylation. In INS-1E cells, suppression of the two main isoforms, PDK1 and PDK3, almost completely prevented PDH E1α phosphorylation. Under basal glucose conditions, phosphorylation was barely detectable and therefore the enzyme almost fully active (90% of maximal). During glucose stimulation, PDH is only partially inhibited (to 78% of maximal). Preventing PDH phosphorylation in situ after suppression of PDK1, 2 and 3 neither enhanced pyruvate oxidation nor insulin secretion. In conclusion, although glucose stimulates E1α phosphorylation and therefore inhibits PDH activity, this control mechanism by itself does not alter metabolism-secretion coupling in INS-1E clonal β-cells.  相似文献   

13.
Heng Zhao  Xianyin Lai  Mu Wang 《Proteomics》2016,16(17):2419-2431
Prostate cancer is one of the leading cancers in men. Taking dietary supplements, such as fish oil (FO), which is rich in n‐3 polyunsaturated fatty acids (PUFAs), has been employed as a strategy to lower prostate cancer risk and control disease progression. In this study, we investigated the global phosphoproteomic changes induced by FO using a combination of phosphoprotein‐enrichment strategy and high‐resolution tandem mass spectrometry. We found that FO induces many more phosphorylation changes than oleic acid when they both are compared to control group. Quantitative comparison between untreated group and FO‐ or oleic acid‐treated groups uncovered a number of important protein phosphorylation changes induced by n‐3PUFAs. This phosphoproteomic discovery study and the follow‐up Western Blot validation study elucidate that phosphorylation levels of the two regulatory serine residues in pyruvate dehydrogenase alpha 1 (PDHA1), serine‐232 and serine‐300, are significantly decreased upon FO treatment. As expected, increased pyruvate dehydrogenase activity was also observed. This study suggests that FO‐induced phosphorylation changes in PDHA1 is more likely related to the glucose metabolism pathway, and n‐3 PUFAs may have a role in controlling the balance between lipid and glucose oxidation.  相似文献   

14.
Starvation for 48 h elicited a 74% increase in hepatic pyruvate dehydrogenase (PDH) kinase activity, measured directly by 32Pi-incorporation from [gamma-32P]ATP into a synthetic peptide corresponding to the major phosphorylation site on E1. The administration of chow ad libitum to previously-starved rats suppressed hepatic PDH kinase activity by only approx. 20% within 2 h of re-feeding, and the relatively high activity of PDH kinase was associated with continued suppression of PDC complex re-activation. Whereas there was no further decline in PDH kinase activity over the next 2 h, PDC re-activation to the fed value was observed during this time interval. PDH kinase activity decreased to fed values only after 8 h.  相似文献   

15.
The present study was designed to identify novel membrane proteins that signal during platelet aggregation. Because one putative mechanism for signaling by a membrane protein involves phosphorylation, we used oligonucleotide-based microarray analyses and mass spectrometric proteomics techniques to specifically discover membrane proteins and also identify those proteins that become phosphorylated on tyrosine, threonine, or serine residues upon platelet aggregation. Surprisingly, both techniques converged to identify a novel membrane protein we have termed PEAR1 (platelet endothelial aggregation receptor 1). Sequence analysis of PEAR1 predicts a type-1 membrane protein, 15 extracellular epidermal growth factor-like repeats, and multiple cytoplasmic tyrosines. Analysis of the tissue distribution of PEAR1 showed that it was most highly expressed in platelets and endothelial cells. Upon platelet aggregation induced by physiological agonists, PEAR1 became phosphorylated on tyrosine (Tyr-925), and serine (Ser-953 and Ser-1029) residues. PEAR1 tyrosine phosphorylation was blocked by eptifibatide, an alpha(IIb)beta(3) antagonist, which inhibits platelet aggregation. Immune clustering of PEAR1 resulted in PEAR1 phosphorylation. Aggregation-induced PEAR1 tyrosine phosphorylation lead to the subsequent association with the ShcB adaptor protein. Platelet proximity induced by centrifugation also induced PEAR1 tyrosine phosphorylation, a reaction not inhibited by eptifibatide. These data suggest that PEAR1 is a novel platelet receptor that signals secondary to alpha(IIb)beta(3)-mediated platelet-platelet contacts.  相似文献   

16.
Pyruvate dehydrogenase (PDH) is a vital regulatory enzyme that catalyzes the conversion of pyruvate into acetyl-CoA and connects anaerobic glycolysis to aerobic TCA cycle. Post-translational inhibition of PDH activity via three serine phosphorylation sites (pS232, pS293, and pS300) regulate the metabolic flux through the TCA cycle, decrease glucose utilization, and facilitate lipid metabolism during times of nutrient deprivation. As metabolic readjustment is necessary to survive hibernation, the purpose of this study was to explore the post-translational regulation of pyruvate dehydrogenase and the expression levels of four mitochondrial serine/threonine kinases (PDHKs), during torpor-arousal cycles in liver, heart, and skeletal muscle of 13-lined ground squirrels. A combination of Luminex multiplex technology and western immunoblotting were used to measure the protein expression levels of total PDH, three phosphorylation sites, S232, 293, 300, and the expression levels of the corresponding PDH kinases (PDHK1-4) during euthermic control, entrance, late torpor, and interbout arousal. Liver and heart showed strong inhibitory PDH regulation, indicating a possible decrease in glucose utilization and a possible preference for β-oxidation of fatty acids during periods of low temperature and starvation. On the contrary, skeletal muscle showed limited PDH regulation via phosphorylation, possibly due to alternate controls. Phosphorylation of PDH may play an important role in regulating aerobic and anaerobic metabolic responses during hibernation in the 13-lined ground squirrel.  相似文献   

17.
A rapid increase in the tyrosine phosphorylation of the non-receptor tyrosine kinase FAK is a prominent early event in fibroblasts stimulated by a variety of signaling molecules. However, a variety of epithelial cells, including intestinal epithelial cells, show a high basal level of tyrosine phosphorylated FAK that is only slightly further increased by addition of G protein-coupled receptor (GPCR) agonists or growth factors. In this study, we determined whether these stimuli could elicit FAK phosphorylation at serine residues, including Ser-910 and Ser-843. Our results show that multiple agonists including angiotensin II (ANGII), lysophosphatidic acid (LPA), phorbol esters and EGF induced a striking stimulation of FAK phosphorylation at Ser-910 in rat intestinal epithelial IEC-18 cells via an ERK-dependent pathway. In striking contrast, none of these stimuli promoted a significant further increase in FAK phosphorylation at Tyr-397 in these cells. These results were extended using cultures of polarized human colonic epithelial T84 cells. We found that either carbachol or EGF promoted a striking ERK-dependent phosphorylation of FAK at Ser-910, but these agonists caused only slight stimulation of FAK at Tyr-397 in T84 cells. In addition, we demonstrated that GPCR agonists also induced a dramatic increase of FAK phosphorylation at Ser-843 in either IEC-18 or T84 cells. Our results indicate that Ser-910 and Ser-843, rather than Tyr-397, are prominent sites differentially phosphorylated in response to neurotransmitters, bioactive lipids, tumor promoters and growth factors in intestinal epithelial cells.  相似文献   

18.
Mutations in the E1alpha subunit gene (PDHA1) of the pyruvate dehydrogenase complex (PDC) are common causes of congenital lactic acidosis. An animal model of E1alpha deficiency could provide insight into the pathological consequences of mutations and serve to test potential therapies. Small interfering RNAs (siRNAs) were designed to cleave the messenger RNA (mRNA) of the E1alpha subunit and were tested in vitro to assess the feasibility of producing a gene knockdown in rats. HEK 293 cells were co-transfected with a rat PDHA1 expression vector and eight naked siRNAs that specifically targeted rat E1alpha mRNA. Quantitative PCR (qPCR) analyses showed that four siRNAs reduced rat PDHA1 RNA levels up to 85% by 24h and up to 65% by 56h, compared to negative and positive controls. Since oligonucleotide-mediated siRNA delivery provided only transient suppression, we next selected two siRNA candidates and generated self-complementary, double-stranded adeno-associated virus (scAAV) vectors (serotypes 2 and 5) expressing a rat short hairpin siRNA expression cassette (scAAVsi-PDHA1). Rat lung fibroblast (RLF) cultures were infected with scAAVsi-PDHA1 vectors. The RLF PDHA1 mRNA level was reduced 53-80% 72h after infection and 54-70% 10 days after infection in RLF cultures. The expression of E1alpha and the specific activity of pyruvate dehydrogenase were also decreased at 10 days after infection in RLF cultures. Thus, scAAV siRNA-mediated knockdown of PDHA1 gene expression provides a strategy that may be applied to create a useful animal model of PDC deficiency.  相似文献   

19.
The four pyruvate dehydrogenase kinase (PDK) and two pyruvate dehydrogenase phosphatase (PDP) isoenzymes that are present in mammalian tissues regulate activity of the pyruvate dehydrogenase complex (PDC) by phosphorylation/dephosphorylation of its pyruvate dehydrogenase (E1) component. The effect of lipoic acids on the activity of PDKs and PDPs was investigated in purified proteins system. R-lipoic acid, S-lipoic acid and R-dihydrolipoic acid did not significantly affect activities of PDPs and at the same time inhibited PDKs to different extents (PDK1?>?PDK4?~?PDK2?>?PDK3 for R-LA). Since lipoic acids inhibited PDKs activity both when reconstituted in PDC and in the presence of E1 alone, dissociation of PDK from the lipoyl domains of dihydrolipoamide acetyltransferase in the presence of lipoic acids is not a likely explanation for inhibition. The activity of PDK1 towards phosphorylation sites 1, 2 and 3 of E1 was decreased to the same extent in the presence of R-lipoic acid, thus excluding protection of the E1 active site by lipoic acid from phosphorylation. R-lipoic acid inhibited autophosphorylation of PDK2 indicating that it exerted its effect on PDKs directly. Inhibition of PDK1 by R-lipoic acid was not altered by ADP but was decreased in the presence of pyruvate which itself inhibits PDKs. An inhibitory effect of lipoic acid on PDKs would result in less phosphorylation of E1 and hence increased PDC activity. This finding provides a possible mechanism for a glucose (and lactate) lowering effect of R-lipoic acid in diabetic subjects.  相似文献   

20.
The four pyruvate dehydrogenase kinase (PDK) and two pyruvate dehydrogenase phosphatase (PDP) isoenzymes that are present in mammalian tissues regulate activity of the pyruvate dehydrogenase complex (PDC) by phosphorylation/dephosphorylation of its pyruvate dehydrogenase (E1) component. The effect of lipoic acids on the activity of PDKs and PDPs was investigated in purified proteins system. R-lipoic acid, S-lipoic acid and R-dihydrolipoic acid did not significantly affect activities of PDPs and at the same time inhibited PDKs to different extents (PDK1>PDK4 approximately PDK2>PDK3 for R-LA). Since lipoic acids inhibited PDKs activity both when reconstituted in PDC and in the presence of E1 alone, dissociation of PDK from the lipoyl domains of dihydrolipoamide acetyltransferase in the presence of lipoic acids is not a likely explanation for inhibition. The activity of PDK1 towards phosphorylation sites 1, 2 and 3 of E1 was decreased to the same extent in the presence of R-lipoic acid, thus excluding protection of the E1 active site by lipoic acid from phosphorylation. R-lipoic acid inhibited autophosphorylation of PDK2 indicating that it exerted its effect on PDKs directly. Inhibition of PDK1 by R-lipoic acid was not altered by ADP but was decreased in the presence of pyruvate which itself inhibits PDKs. An inhibitory effect of lipoic acid on PDKs would result in less phosphorylation of E1 and hence increased PDC activity. This finding provides a possible mechanism for a glucose (and lactate) lowering effect of R-lipoic acid in diabetic subjects.  相似文献   

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