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Five enzymes of the glycolytic pathway serve as substrates for purified epidermal-growth-factor-receptor kinase.

ABSTRACT: Several enzymes of the glycolytic pathway are phosphorylated in vitro and in vivo by retroviral transforming protein kinases. These substrates include the enzymes phosphoglycerate mutase (PGM), enolase and lactate dehydrogenase (LDH). Here we show that purified EGF (epidermal growth factor)-receptor kinase phosphorylates the enzymes PGM and enolase and also the key regulatory enzymes of the glycolytic pathway, phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in an EGF-dependent manner. Stoichiometry of phosphate incorporation into GAPDH (calculated from native Mr) is the highest, reaching approximately 1. LDH and other enzymes of the glycolytic pathway are not phosphorylated by the purified EGF-receptor kinase. These enzymes are phosphorylated under native conditions, and the Km values of EGF-receptor kinase for their phosphorylation are close to the physiological concentrations of these enzymes in the cell. EGF stimulates the reaction by 2-5-fold by increasing the Vmax. without affecting the Km of this process. Phosphorylation is rapid at 22 degrees C and at higher temperatures. However, unlike the self-phosphorylation of EGF-receptor, which occurs at 4 degrees C, the glycolytic enzymes are poorly phosphorylated at this temperature. Some enzymes, in particular enolase, increase the receptor Km for ATP in the autophosphorylation process and thus may act as competitive inhibitors of EGF-receptor self-phosphorylation. On the basis of the Km values of EGF receptor for the substrate enzymes and for ATP in the phosphorylation reaction, these enzymes may also be substrates in vivo for the EGF-receptor kinase.

SUBMITTER: Reiss N

PROVIDER: S-EPMC1147341 | biostudies-other | 1986 Nov

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:Spleen tyrosine kinase (SYK) is frequently upregulated in recurrent ovarian carcinomas, for which effective therapy is urgently needed. SYK phosphorylates several substrates, but their translational implications remain unclear. Here, we show that SYK interacts with EGFR and ERBB2, and directly enhances their phosphorylation. METHODS:We used immunohistochemistry and immunoblotting to assess SYK and EGFR phosphorylation in ovarian serous carcinomas. Association with survival was determined by Kaplan-Meier analysis and the log-rank test. To study its role in EGFR signaling, SYK activity was modulated using a small molecule inhibitor, a syngeneic knockout, and an active kinase inducible system. We applied RNA-seq and phosphoproteomic mass spectrometry to investigate the SYK-regulated EGF-induced transcriptome and downstream substrates. FINDINGS:Induced expression of constitutively active SYK130E reduced cellular response to EGFR/ERBB2 inhibitor, lapatinib. Expression of EGFRWT, but not SYK non-phosphorylatable EGFR3F mutant, resulted in paclitaxel resistance, a phenotype characteristic to SYK active ovarian cancers. In tumor xenografts, SYK inhibitor reduces phosphorylation of EGFR substrates. Compared to SYKWT cells, SYKKO cells have an attenuated EGFR/ERBB2-transcriptional activity and responsiveness to EGF-induced transcription. In ovarian cancer tissues, pSYK (Y525/526) levels showed a positive correlation with pEGFR (Y1187). Intense immunoreactivity of pSYK (Y525/526) correlated with poor overall survival in ovarian cancer patients. INTERPRETATION:These findings indicate that SYK activity positively modulates the EGFR pathway, providing a biological foundation for co-targeting SYK and EGFR. FUND: Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine, NIH/NCI, Ovarian Cancer Research Foundation Alliance, HERA Women's Cancer Foundation and Roseman Foundation. Funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript and eventually in the decision to submit the manuscript.

Project description:BACKGROUND:Novel therapeutic approaches are required to treat ovarian cancer and dependency on glycolysis may provide new targets for treatment. This study sought to investigate the variation of expression of molecular components (GLUT1, HKII, PKM2, LDHA) of the glycolytic pathway in ovarian cancers and the effectiveness of targeting this pathway in ovarian cancer cell lines with inhibitors. METHODS:Expression of GLUT1, HKII, PKM2, LDHA were analysed by quantitative immunofluorescence in a tissue microarray (TMA) analysis of 380 ovarian cancers and associations with clinicopathological features were sought. The effect of glycolysis pathway inhibitors on the growth of a panel of ovarian cancer cell lines was assessed by use of the SRB proliferation assay. Combination studies were undertaken combining these inhibitors with cytotoxic agents. RESULTS:Mean expression levels of GLUT1 and HKII were higher in high grade serous ovarian cancer (HGSOC), the most frequently occurring subtype, than in non-HGSOC. GLUT1 expression was also significantly higher in advanced stage (III/IV) ovarian cancer than early stage (I/II) disease. Growth dependency of ovarian cancer cells on glucose was demonstrated in a panel of ovarian cancer cell lines. Inhibitors of the glycolytic pathway (STF31, IOM-1190, 3PO and oxamic acid) attenuated cell proliferation in platinum-sensitive and platinum-resistant HGSOC cell line models in a concentration dependent manner. In combination with either cisplatin or paclitaxel, 3PO (a novel PFKFB3 inhibitor) enhanced the cytotoxic effect in both platinum sensitive and platinum resistant ovarian cancer cells. Furthermore, synergy was identified between STF31 (a novel GLUT1 inhibitor) or oxamic acid (an LDH inhibitor) when combined with metformin, an inhibitor of oxidative phosphorylation, resulting in marked inhibition of ovarian cancer cell growth. CONCLUSIONS:The findings of this study provide further support for targeting the glycolytic pathway in ovarian cancer and several useful combinations were identified.

Project description:ObjectiveElevated glycolytic enzymes in renal glomeruli correlated with preservation of renal function in the Medalist Study, individuals with ≥50 years of type 1 diabetes. Specifically, pyruvate kinase M2 (PKM2) activation protected insulin-deficient diabetic mice from hyperglycemia-induced glomerular pathology. This study aims to extend these findings in a separate cohort of individuals with type 1 and type 2 diabetes and discover new circulatory biomarkers for renal protection through proteomics and metabolomics of Medalists' plasma. We hypothesize that increased glycolytic flux and improved mitochondrial biogenesis will halt the progression of diabetic nephropathy.Research design and methodsImmunoblots analyzed selected glycolytic and mitochondrial enzymes in postmortem glomeruli of non-Medalists with type 1 diabetes (n = 15), type 2 diabetes (n = 19), and no diabetes (n = 5). Plasma proteomic (SOMAscan) (n = 180) and metabolomic screens (n = 214) of Medalists with and without stage 3b chronic kidney disease (CKD) were conducted and significant markers validated by ELISA.ResultsGlycolytic (PKM1, PKM2, and ENO1) and mitochondrial (MTCO2) enzymes were significantly elevated in glomeruli of CKD- versus CKD+ individuals with type 2 diabetes. Medalists' plasma PKM2 correlated with estimated glomerular filtration rate (r 2 = 0.077; P = 0.0002). Several glucose and mitochondrial enzymes in circulation were upregulated with corresponding downregulation of toxic metabolites in CKD-protected Medalists. Amyloid precursor protein was also significantly upregulated, tumor necrosis factor receptors downregulated, and both confirmed by ELISA.ConclusionsElevation of enzymes involved in the metabolism of intracellular free glucose and its metabolites in renal glomeruli is connected to preserving kidney function in both type 1 and type 2 diabetes. The renal profile of elevated glycolytic enzymes and reduced toxic glucose metabolites is reflected in the circulation, supporting their use as biomarkers for endogenous renal protective factors in people with diabetes.

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Five enzymes of the glycolytic pathway serve as substrates for purified epidermal-growth-factor-receptor kinase.

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