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The RIPE3b1 activator of the insulin gene is composed of a protein(s) of approximately 43 kDa, whose DNA binding activity is inhibited by protein phosphatase treatment.

Zhao L, Cissell MA, Henderson E, Colbran R, Stein R
J Biol Chem. 2000 275 (14): 10532-7

PMID: 10744746 · DOI:10.1074/jbc.275.14.10532

Glucose-stimulated and pancreatic islet beta cell-specific expression of the insulin gene is mediated in part by the C1 DNA-element binding complex, termed RIPE3b1. In this report, we define the molecular weight range of the protein(s) that compose this beta cell-enriched activator complex and show that protein phosphatase treatment inhibits RIPE3b1 DNA binding activity. Fractionation of beta cell nuclear extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that RIPE3b1 binding was mediated by a protein(s) within the 37-49-kDa ranges. Direct analysis of the proteins within the RIPE3b1 complex by ultraviolet light cross-linking analysis identified three binding species of approximately 51, 45, and 38 kDa. Incubating beta cell nuclear extracts with either calf alkaline phosphatase or a rat brain phosphatase preparation dramatically reduced RIPE3b1 DNA complex formation. Phosphatase inhibition of RIPE3b1 binding was prevented by sodium pyrophosphate, a general phosphatase inhibitor. We discuss how changes in the phosphorylation status of the RIPE3b1 activator may influence its DNA binding activity.

MeSH Terms (19)

3T3 Cells Animals Cell Line Cell Nucleus Cricetinae Enhancer Elements, Genetic Homeodomain Proteins Insulin Islets of Langerhans Liver Neoplasms, Experimental Mice Molecular Weight Mutagenesis, Site-Directed Phosphoprotein Phosphatases Point Mutation Promoter Regions, Genetic Rats Trans-Activators Tumor Cells, Cultured

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