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J Biol Chem, Vol. 275, Issue 14, 10532-10537, April 7, 2000

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^*

Li Zhao, Michelle A. Cissell, Eva Henderson, Roger Colbran, and Roland Stein

From the Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232

Glucose-stimulated and pancreatic islet  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  cell-enriched activator complex and show that protein phosphatase treatment inhibits RIPE3b1 DNA binding activity. Fractionation of  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  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.

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^* This work was supported by National Institutes of Health Grants DK49852, DK42502 (to R. S.), and NS37508 (to R. C.) and partial support from the Vanderbilt University Diabetes Research and Training Center Molecular Biology Core Laboratory (Public Health Service Grant P60 DK20593 from the National Institutes of Health).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 615-322-7026; Fax: 615-322-7236; E-mail: Roland.Stein@mcmail.vanderbilt.edu.

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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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