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 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.
Zhao
L
L
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
Cissell
M A
MA
Henderson
E
E
Colbran
R
R
Stein
R
R
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D004742:Enhancer Elements, Genetic; D018398:Homeodomain Proteins / Q000737:chemistry / Q000378:metabolism; D007328:Insulin / Q000235:genetics*; D007515:Islets of Langerhans / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental; D051379:Mice; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D051381:Rats; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D018398:Homeodomain Proteins; D007328:Insulin; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D010749:Phosphoprotein Phosphatases