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
L. Zhao et al., 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, J BIOL CHEM, 275(14), 2000, pp. 10532-10537
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.