Epidermal Growth Factor and Okadaic Acid Stimulate Sp1 Proteolysis

Sp1 nuclear levels have been shown todirectly correlate with the proliferative state of the cell. We therefore studied changes in the abundance of Sp1 in a rat pituitary cell line GH4 whose growth rate is regulated by epidermal growth factor (EGF). Nuclear extracts from GH4cells treated with 10 nm EGF for at least 16 h showed a 50% decrease in Sp1 binding to a GC-rich element present in the gastrin promoter. The decrease in binding correlated with a decrease in cell proliferation, a loss of nuclear Sp1 protein and a 50–60% decrease in Sp1-mediated transactivation through an Sp1 enhancer element in transfection assays. Okadaic acid, a phosphatase inhibitor, was synergistic with the effect of EGF on Sp1 protein levels suggesting that the loss of Sp1 was mediated by phosphorylation events. This result was confirmed by showing a 2-fold increase in orthophosphate-labeled Sp1 with EGF and okadaic acid. Cycloheximide prevented the expected loss of Sp1 mediated by EGF and okadaic acid suggesting that the synthesis of a protease may mediate these events. This hypothesis was tested directly by showing that the cysteine protease inhibitor leupeptin prevented Sp1 degradation. Using the PEST-FIND computer program, the computed PEST score for human and rat Sp1 is 10.4 and 13.7, respectively, indicating that Sp1 has a domain with a high concentration of proline, glutamic acid, serine, and threonine residues as reported for a number of proteins with inducible rates of degradation. Collectively, these results indicate that sustained stimulation of GH4 cells by EGF initiates a cascade of phosphorylation events that promotes Sp1 proteolysis, decreased Sp1 nuclear levels and decreased cellular proliferation.