EPIDERMAL GROWTH-FACTOR AND OKADAIC ACID STIMULATE SP1 PROTEOLYSIS

Sp1 nuclear levels have been shown to directly correlate with the prol iferative state of the cell. We therefore studied changes in the abund ance of Sp1 in a rat pituitary cell line GH(4) whose growth rate is re gulated by epidermal growth factor (EGF). Nuclear extracts from GH, ce lls 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 tr ansactivation 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 medi ated by phosphorylation events. This result was confirmed by showing a 2-fold increase in orthophosphate-labeled Sp1 with EGF and okadaic ac id, Cycloheximide prevented the expected loss of Sp1 mediated by EGF a nd okadaic acid suggesting that the synthesis of a protease may mediat e these events. This hypothesis was tested directly by showing that th e cysteine protease inhibitor leupeptin prevented Sp1 degradation. Usi ng the PEST-FIND computer program, the computed PEST score for human a nd rat Sp1 is 10.4 and 13.7, respectively, indicating that Sp1 has a d omain with a high concentration of proline, glutamic acid, serine, and threonine residues as reported for a number of proteins with inducibl e rates of degradation. Collectively, these results indicate that sust ained stimulation of GH(4) cells by EGF initiates a cascade of phospho rylation events that promotes Sp1 proteolysis, decreased Sp1 nuclear l evels and decreased cellular proliferation.