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

Glucose-stimulated and pancreatic islet beta cell-specific expression of th e insulin gene is mediated in part by the C1 DNA-element binding complex, t ermed 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. F ractionation of beta cell nuclear extracts by sodium dodecyl sulfate-polyac rylamide 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 iden tified three binding species of approximately 51, 45, and 38 kDa. Incubatin g beta cell nuclear extracts with either calf alkaline phosphatase or a rat brain phosphatase preparation dramatically reduced RIPE3b1 DNA complex for mation. Phosphatase inhibition of RIPE3b1 binding was prevented by sodium p yrophosphate, a general phosphatase inhibitor. We discuss how changes in th e phosphorylation status of the RIPE3b1 activator may influence its DNA bin ding activity.