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1.
PLoS One ; 9(3): e90687, 2014.
Article in English | MEDLINE | ID: mdl-24595027

ABSTRACT

Extracellular fibroblast growth factor 1 (FGF1) acts through cell surface tyrosine kinase receptors, but FGF1 can also act directly in the cell nucleus, as a result of nuclear import of endogenously produced, non-secreted FGF1 or by transport of extracellular FGF1 via endosomes and cytosol into the nucleus. In the nucleus, FGF1 can be phosphorylated by protein kinase C δ (PKCδ), and this event induces nuclear export of FGF1. To identify intracellular targets of FGF1 we performed affinity pull-down assays and identified nucleolin, a nuclear multifunctional protein, as an interaction partner of FGF1. We confirmed a direct nucleolin-FGF1 interaction by surface plasmon resonance and identified residues of FGF1 involved in the binding to be located within the heparin binding site. To assess the biological role of the nucleolin-FGF1 interaction, we studied the intracellular trafficking of FGF1. In nucleolin depleted cells, exogenous FGF1 was endocytosed and translocated to the cytosol and nucleus, but FGF1 was not phosphorylated by PKCδ or exported from the nucleus. Using FGF1 mutants with reduced binding to nucleolin and a FGF1-phosphomimetic mutant, we showed that the nucleolin-FGF1 interaction is critical for the intranuclear phosphorylation of FGF1 by PKCδ and thereby the regulation of nuclear export of FGF1.


Subject(s)
Cell Nucleus/metabolism , Fibroblast Growth Factor 1/metabolism , Phosphoproteins/metabolism , RNA-Binding Proteins/metabolism , Active Transport, Cell Nucleus , Animals , Cell Line , Fibroblast Growth Factor 1/analysis , Fibroblast Growth Factor 2/metabolism , HEK293 Cells , Humans , Mice , NIH 3T3 Cells , Phosphorylation , Nucleolin
2.
J Biol Chem ; 285(29): 22403-13, 2010 Jul 16.
Article in English | MEDLINE | ID: mdl-20452975

ABSTRACT

Phosphatidylcholine (PC) is synthesized from choline via the CDP-choline pathway. Liver cells can also synthesize PC via the sequential methylation of phosphatidylethanolamine, catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). The current study investigates whether or not hepatic PC biosynthesis is linked to diet-induced obesity. Pemt(+/+) mice fed a high fat diet for 10 weeks increased in body mass by 60% and displayed insulin resistance, whereas Pemt(-/-) mice did not. Compared with Pemt(+/+) mice, Pemt(-/-) mice had increased energy expenditure and maintained normal peripheral insulin sensitivity; however, they developed hepatomegaly and steatosis. In contrast, mice with impaired biosynthesis of PC via the CDP-choline pathway in liver became obese when fed a high fat diet. We, therefore, hypothesized that insufficient choline, rather than decreased hepatic phosphatidylcholine, was responsible for the lack of weight gain in Pemt(-/-) mice despite the presence of 1.3 g of choline/kg high fat diet. Supplementation with an additional 2.7 g of choline (but not betaine)/kg of diet normalized energy metabolism, weight gain, and insulin resistance in high fat diet-fed Pemt(-/-) mice. Furthermore, Pemt(+/+) mice that were fed a choline-deficient diet had increased oxygen consumption, had improved glucose tolerance, and gained less weight. Thus, de novo synthesis of choline via PEMT has a previously unappreciated role in regulating whole body energy metabolism.


Subject(s)
Choline/biosynthesis , Diet , Obesity/enzymology , Obesity/prevention & control , Phosphatidylethanolamine N-Methyltransferase/deficiency , Animals , Betaine/administration & dosage , Betaine/pharmacology , Dietary Fats/administration & dosage , Dietary Fats/pharmacology , Dietary Supplements , Energy Metabolism/drug effects , Fatty Liver/chemically induced , Fatty Liver/complications , Fatty Liver/enzymology , Fatty Liver/pathology , Feeding Behavior/drug effects , Insulin Resistance , Male , Metabolic Networks and Pathways/drug effects , Mice , Mice, Inbred C57BL , Mitochondria/drug effects , Mitochondria/ultrastructure , Obesity/chemically induced , Obesity/complications , Phenotype , Phosphatidylcholines/biosynthesis , Phosphatidylethanolamine N-Methyltransferase/metabolism , Weight Gain/drug effects
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