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1.
Mol Cell Biol ; 23(3): 852-63, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12529391

ABSTRACT

The ribosomal protein S6 kinase (S6K) belongs to the AGC family of Ser/Thr kinases and is known to be involved in the regulation of protein synthesis and the G(1)/S transition of the cell cycle. There are two forms of S6K, termed S6Kalpha and S6Kbeta, which have cytoplasmic and nuclear splice variants. Nucleocytoplasmic shuttling has been recently proposed for S6Kalpha, based on the use of the nuclear export inhibitor, leptomycin B. However, the molecular mechanisms regulating subcellular localization of S6Ks in response to mitogenic stimuli remain to be elucidated. Here we present data on the in vitro and in vivo phosphorylation of S6Kbeta, but not S6Kalpha, by protein kinase C (PKC). The site of phosphorylation was identified as S486, which is located within the C-terminal nuclear localization signal. Mutational analysis and the use of phosphospecific antibodies provided evidence that PKC-mediated phosphorylation at S486 does not affect S6K activity but eliminates the function of its nuclear localization signal and causes retention of an activated form of the kinase in the cytoplasm. Taken together, this study uncovers a novel mechanism for the regulation of nucleocytoplasmic shuttling of S6KbetaII by PKC-mediated phosphorylation.


Subject(s)
Protein Kinase C/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Active Transport, Cell Nucleus , Amino Acid Sequence , Binding Sites , Cell Line , Fatty Acids, Unsaturated/pharmacology , Humans , In Vitro Techniques , Insulin/pharmacology , Isoenzymes/metabolism , Mitogens/pharmacology , Molecular Sequence Data , Phenylephrine/pharmacology , Phosphorylation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/chemistry , Ribosomal Protein S6 Kinases, 70-kDa/genetics , Signal Transduction , Subcellular Fractions/enzymology , Tetradecanoylphorbol Acetate/pharmacology , Transfection
2.
J Biol Chem ; 278(50): 50316-21, 2003 Dec 12.
Article in English | MEDLINE | ID: mdl-14514684

ABSTRACT

CoA synthase mediates the last two steps in the sequence of enzymatic reactions, leading to CoA biosynthesis. We have recently identified cDNA for CoA synthase and demonstrated that it encodes a bifunctional enzyme possessing 4'-phosphopantetheine adenylyltransferase and dephospho-CoA kinase activities. Molecular cloning of CoA synthase provided us with necessary tools to study subcellular localization and the regulation of this bifunctional enzyme. Transient expression studies and confocal microscopy allowed us to demonstrate that full-length CoA synthase is associated with the mitochondria, whereas the removal of the N-terminal region relocates the enzyme to the cytosol. In addition, we showed that the N-terminal sequence of CoA synthase (amino acids 1-29) exhibits a hydrophobic profile and targets green fluorescent protein exclusively to mitochondria. Further analysis, involving subcellular fractionation and limited proteolysis, indicated that CoA synthase is localized on the mitochondrial outer membrane. Moreover, we demonstrate for the first time that phosphatidylcholine and phosphatidylethanolamine, which are the main components of the mitochondrial outer membrane, are potent activators of both enzymatic activities of CoA synthase in vitro. Taken together, these data provide the evidence that the final stages of CoA biosynthesis take place on mitochondria and the activity of CoA synthase is regulated by phospholipids.


Subject(s)
Coenzyme A Ligases/biosynthesis , Coenzyme A Ligases/genetics , Gene Expression Regulation, Enzymologic , Amino Acid Sequence , Animals , Cell Line , Cell Line, Tumor , Cell Membrane/metabolism , Centrifugation, Density Gradient , Cloning, Molecular , Coenzyme A/metabolism , DNA, Complementary/metabolism , Green Fluorescent Proteins , Humans , Luminescent Proteins/metabolism , Mice , Microscopy, Confocal , Microscopy, Fluorescence , Mitochondria/metabolism , Molecular Sequence Data , NIH 3T3 Cells , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/genetics , Phospholipids/chemistry , Plasmids/metabolism , Protein Structure, Tertiary , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Subcellular Fractions/metabolism
3.
J Biol Chem ; 277(25): 22107-10, 2002 Jun 21.
Article in English | MEDLINE | ID: mdl-11980892

ABSTRACT

Coenzyme A functions as a carrier of acetyl and acyl groups in living cells and is essential for numerous biosynthetic, energy-yielding, and degradative metabolic pathways. There are five enzymatic steps in CoA biosynthesis. To date, molecular cloning of enzymes involved in the CoA biosynthetic pathway in mammals has been only reported for pantothenate kinase. In this study, we present cDNA cloning and functional characterization of CoA synthase. It has an open reading frame of 563 aa and encodes a protein of approximately 60 kDa. Sequence alignments suggested that the protein possesses both phosphopantetheine adenylyltransferase and dephospho-CoA kinase domains. Biochemical assays using wild type recombinant protein confirmed the gene product indeed contained both these enzymatic activities. The presence of intrinsic phosphopantetheine adenylyltransferase activity was further confirmed by site-directed mutagenesis. Therefore, this study describes the first cloning and characterization of a mammalian CoA synthase and confirms this is a bifunctional enzyme containing the last two components of CoA biosynthesis.


Subject(s)
Acetate-CoA Ligase/genetics , Coenzyme A/biosynthesis , Transferases/genetics , Amino Acid Sequence , Animals , Blotting, Northern , Cell Line , Cloning, Molecular , DNA, Complementary/metabolism , Humans , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Plasmids/metabolism , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Species Specificity , Tissue Distribution , Two-Hybrid System Techniques
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