Abstract
Insulin-like growth factor binding protein-4, Miz-1, leptin, prostaglandin D synthase, and granulin precursor were identified as proteins interacting with the N-terminal half of mammalian Type III hexokinase (HKIII) in the yeast two-hybrid method. These interactions were confirmed by in vitro binding studies. All five of these proteins, and their mRNAs, were present in PC12 cells, as shown by immunoblotting and RT-PCR, respectively. All were coimmunoprecipitated from PC12 extracts with an antibody against HKIII, but not with anti-Type I hexokinase. Moreover, all of these proteins were coimmunoprecipitated using antileptin as precipitating antibody, indicating the existence of a macromolecular complex including these five proteins and HKIII. Transfection of M+R 42 cells with HKIII-green fluorescent protein (GFP) reporter constructs gave a diffuse intracellular fluorescence. Cotransfection with leptin or Miz-1 resulted in distinctly different localization of the HKIII-GFP fusion protein, at intracellular sites coincident with localization of leptin-GFP or Miz-1-GFP reporter constructs.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Base Sequence
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Binding Sites
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DNA Primers / genetics
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DNA-Binding Proteins / metabolism
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Glycoproteins / metabolism
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Green Fluorescent Proteins
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Growth Substances / metabolism
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Hexokinase / chemistry*
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Hexokinase / genetics
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Hexokinase / metabolism*
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In Vitro Techniques
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Insulin-Like Growth Factor Binding Protein 4 / metabolism
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Intercellular Signaling Peptides and Proteins*
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Intramolecular Oxidoreductases / metabolism
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Leptin / metabolism
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Lipocalins
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Luminescent Proteins / chemistry
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism
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PC12 Cells
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Progranulins
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Protein Binding
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Rats
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Saccharomyces cerevisiae / genetics
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Transfection
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Two-Hybrid System Techniques
Substances
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DNA Primers
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DNA-Binding Proteins
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Glycoproteins
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Growth Substances
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Insulin-Like Growth Factor Binding Protein 4
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Intercellular Signaling Peptides and Proteins
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Leptin
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Lipocalins
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Luminescent Proteins
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Progranulins
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Recombinant Fusion Proteins
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Green Fluorescent Proteins
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Hexokinase
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Intramolecular Oxidoreductases
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prostaglandin R2 D-isomerase