Dimeric and tetrameric forms of muscle fructose-1,6- bisphosphatase play different roles in the cell

Muscle fructose 1,6-bisphosphatase (FBP2), besides being a regulatory enzyme of glyconeogenesis also protects mitochondria against calcium stress and plays a key role in regulation of the cell cycle, promoting cardiomyocytes survival. However, in cancer cells, FBP2 acts as an anti-oncogenic/anti-proliferative protein. Here, we show that the physiological function of FBP2 depends both on its level of expression in a cell as well as its oligomerization state. Animal fructose-1,6-bisphosphatases are thought to function as tetramers. We present evidence that FBP2 exists in an equilibrium between tetramers and dimers. The dimeric form is fully active and insensitive to AMP, the main allosteric inhibitor of FBP2. Tetramerization induces the sensitivity of the protein to AMP, but it requires the presence of a hydrophobic central region in which leucine 190 plays a crucial role. Only the tetrameric form of FBP2 is retained in cardiomyocyte cell nucleus whereas only the dimeric form associates with mitochondria and protects them against stress stimuli, such as elevated calcium and H2O2 level. Remarkably, in hypoxic conditions, which are typical for many cancers, FBP2 ceases to interact with mitochondria and loses its pro-survival potential. Our results throw new light on the basis of the diverse role of FBP2 in cells

Neuron-derived transthyretin modulates astrocytic glycolysis in hormone-independent manner

It has been shown that neurons alter the expression of astrocytic metabolic enzymes by secretion of until now unknown molecule(s) into extracellular fluid. Here, we present evidence that neuron-derived transthyretin (TTR) stimulates expression of glycolytic enzymes in astrocytes which is reflected by an increased synthesis of ATP. The action of TTR is restricted to regulatory enzymes of glycolysis: phosphofructokinase P (PFKP) and pyruvate kinase M1/M2 isoforms (PKM1/2). The regulation of PFK and PKM expression by TTR is presumably specific for brain tissue and is independent of the role of TTR as a carrier protein for thyroxine and retinol. TTR induced expression of PKM and PFK is mediated by the cAMP/PKA-dependent pathway and is antagonized by the PI3K/Akt pathway. Our results provide the first experimental evidence for action of TTR as a neuron-derived energy metabolism activator in astrocytes and describe the mechanisms of its action. The data presented here suggest that TTR is involved in a mechanism in which neurons stimulate degradation of glycogen-derived glucosyl units without significant modulation of glucose uptake by glial cells

A potential role for muscle in glucose homeostasis: in vivo kinetic studies in glycogen storage disease type 1a and fructose-1,6-bisphosphatase deficiency

A potential role for muscle in glucose homeostasis was recently suggested based on characterization of extrahepatic and extrarenal glucose-6-phosphatase (glucose-6-phosphatase-beta). To study the role of extrahepatic tissue in glucose homeostasis during fasting glucose kinetics were studied in two patients with a deficient hepatic and renal glycogenolysis and/or gluconeogenesis. Endogenous glucose production (EGP), glycogenolysis (GGL), and gluconeogenesis (GNG) were quantified with stable isotopes in a patient with glycogen storage disease type 1a (GSD-1a) and a patient with fructose-1,6-bisphosphatase (FBPase) deficiency. The [6,6-H-2(2)]glucose dilution method in combination with the deuterated water method was used during individualized fasting tests. Both patients became hypoglycemic after 2.5 and 14.5 h fasting, respectively. At that time, the patient with GSD-1a had EGP 3.84 mu mol/kg per min (30% of normal EGP after an overnight fast), GGL 3.09 mu mol/kg per min, and GNG 0.75 mu mol/kg per min. The patient with FBPase deficiency had EGP 8.53 mu mol/kg per min (62% of normal EGP after an overnight fast), GGL 6.89 mu mol/kg per min GGL, and GNG 1.64 mu mol/kg per min. EGP was severely hampered in both patients, resulting in hypoglycemia. However, despite defective hepatic and renal GNG in both disorders and defective hepatic GGL in GSD-1a, both patients were still able to produce glucose via both pathways. As all necessary enzymes of these pathways have now been functionally detected in muscle, a contribution of muscle to EGP during fasting via both GGL as well as GNG is suggeste

Alteration of AKT Activity Increases Chemotherapeutic Drug and Hormonal Resistance in Breast Cancer yet Confers an Achilles Heel by Sensitization to Targeted Therapy

The PI3K/PTEN/Akt/mTOR pathway plays critical roles in the regulation of cell growth. The effects of this pathway on drug resistance and cellular senescence of breast cancer cells has been a focus of our laboratory. Introduction of activated Akt or mutant PTEN constructs which lack lipid phosphatase [PTEN(G129E)] or lipid and protein phosphatase [PTEN(C124S)] activity increased the resistance of the cells to the chemotherapeutic drug doxorubicin, and the hormonal drug tamoxifen. Activated Akt and PTEN genes also inhibited the induction of senescence after doxorubicin treatment; a phenomenon associated with unrestrained proliferation and tumorigenesis. Interference with the lipid phosphatase domain of PTEN was sufficient to activate Akt/mTOR/p70S6K as MCF-7 cells transfected with the mutant PTEN gene lacking the lipid phosphatase activity [PTEN(G129E)] displayed elevated levels of activated Akt and p70S6K compared to empty vector transfected cells. Cells transfected with mutant PTEN or Akt constructs were hypersensitive to mTOR inhibitors when compared with the parental or empty vector transfected cells. Akt-transfected cells were cultured for over two months in tamoxifen from which tamoxifen and doxorubicin resistant cells were isolated that were >10-fold more resistant to tamoxifen and doxorubicin than the original Akt-transfected cells. These cells had a decreased induction of both activated p53 and total p21Cip1 upon doxorubicin treatment. Furthermore, these cells had an increased inactivation of GSK-3β and decreased expression of the estrogen receptor-α. In these drug resistant cells, there was an increased activation of ERK which is associated with proliferation. These drug resistant cells were hypersensitive to mTOR inhibitors and also sensitive to MEK inhibitors, indicating that the enhanced p70S6K and ERK expression was relevant to their drug and hormonal resistance. Given that Akt is overexpressed in greater than 50% of breast cancers, our results point to potential therapeutic targets, mTOR and MEK. These studies indicate that activation of the Akt kinase or disruption of the normal activity of the PTEN phosphatase can have dramatic effects on activity of p70S6K and other downstream substrates and thereby altering the therapeutic sensitivity of breast cancer cells. The effects of doxorubicin and tamoxifen on induction of the Raf/MEK/ERK and PI3K/Akt survival pathways were examined in unmodified MCF-7 breast cells. Doxorubicin was a potent inducer of activated ERK and to a lesser extent Akt. Tamoxifen also induced ERK. Thus a consequence of doxorubicin and tamoxifen therapy of breast cancer is the induction of a pro-survival pathway which may contribute to the development of drug resistance. Unmodified MCF-7 cells were also sensitive to MEK and mTOR inhibitors which synergized with both tamoxifen and doxorubicin to induce death. In summary, our results point to the key interactions between the PI3K/PTEN/Akt/mTOR and Raf/ MEK/ERK pathways in regulating chemotherapeutic drug resistance/sensitivity in breast cancer and indicate that targeting these pathways may prevent drug and hormonal resistance. Orignally published Advances in Enzyme Regulation, Vol. 48, No. 1, 2008

Aldolase A is present in smooth muscle cell nuclei

Previously we have shown that aldolase (ALD; EC 4.1.2.13) is present in cardiomyocyte nuclei. Now, we focused our attention on ALD localization in smooth muscle cells. Immunocytochemical methods were used to study the subcellular localization of ALD. Aldolase was localized in the cytoplasm as well as in the nuclei. Within the nuclei ALD was located in the heterochromatin region. Native polyacrylamide gel electrophoresis followed by aldolase activity staining in gel was used to study the ALD isoenzyme pattern in porcine smooth muscle cells. Two ALD isoenzymes, A and C, were found in these cells but in the nuclei only the muscle isoenzyme was detected. To support the nuclear localization of ALD, measurement of aldolase activity in the smooth muscle cell nuclei isolated from porcine stomach was performed. The ALD activity in the isolated nuclei was detectable only after preincubation of the nuclear fraction with Triton X-100 and high concentration of KCl

Rabbit muscle fructose-1,6-bisphosphatase is phosphorylated in vivo.

Phosphorylated fructose-1,6-bisphosphatase (FBPase) was isolated from rabbit muscle in an SDS/PAGE homogeneous form. Its dephosphorylation with alkaline phosphatase revealed 2.8 moles of inorganic phosphate per mole of FBPase. The phosphorylated FBPase (P-FBPase) differs from the dephosphorylated enzyme in terms of its kinetic properties like Km and kcat, which are two times higher for the phosphorylated FBPase, and in the affinity for aldolase, which is three times lower for the dephosphorylated enzyme. ephosphorylated FBPase can be a substrate for protein kinase A and the amount of phosphate incorporated per FBPase monomer can reach 2-3 molecules. Since interaction of muscle aldolase with muscle FBPase results in desensitisation of the latter toward AMP inhibition (Rakus & Dzugaj, 2000, Biochem. Biophys. Res. Commun. 275, 611-616), phosphorylation may be considered as a way of muscle FBPase activity regulation

Human lung fructose-1,6-bisphosphatase is localized in pneumocytes II

The localization of fructose-1,6-bisphosphatase (Fru-1,6-Pase EC 3.1.3.11) in human alveolar epithelium was determined immunohistochemically using a polyclonal antibody raised against the enzyme purified from human liver. The immunohistochemical analysis revealed that the Fru-1,6-Pase was localized in pneumocytes 11 and was absent in pneumocytes 1. Hypothetically Fru-1,6-Pase participating in glucose-6-phosphate synthesis from noncarbohydrate precursors increases NADPH leve1 which is used for surfactant synthesis and for glutathione redox cycle

Nuclear localization of aldolase A in pig cardiomyocytes

The subcellular localization of the muscle aldolase (aldolase A) in cardiomyocytes was determined immunocytochemically by light and electron microscopy. The enzyme was localized in the cytoplasm and also in cardiomyocyte nuclei. Inside the nuclei it was preferentially localized in the heterochromatin region. The nuclear localization was confirmed by the measurement of aldolase activity in subcellular fractions of a heart muscle, and in isolated nuclei of cardiomyocytes. There was no detectable aldolase activity in isolated cardiomyocyte nuclei fractions if the fraction was not preincubated with a solution containing Triton X-100 and KCl. The calculated concentration of aldolase in the nucleus was about 0.6 µM. This paper is the first report on the localization of aldolase A inside cardiomyocyte nuclei