Epidermal growth factor and okadaic acid stimulate Sp1 proteolysis

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Sp1 nuclear levels correlate with cellular proliferation.
• Epidermal growth factor (EGF) regulates the growth rate of GH4 rat pituitary cells.

Purpose of the Study:

• To investigate the effect of EGF on Sp1 abundance and activity in GH4 cells.
• To elucidate the molecular mechanisms underlying EGF-mediated changes in Sp1.

Main Methods:

• GH4 cells were treated with EGF and analyzed for Sp1 binding, nuclear protein levels, and transactivation activity.
• Phosphorylation events were assessed using orthophosphate labeling.
• Proteolysis was investigated using cycloheximide and protease inhibitors (leupeptin).
• PEST-FIND program was used to analyze Sp1 for degradation motifs.

Main Results:

• EGF treatment (10 nM, ≥16 h) decreased Sp1 binding to the gastrin promoter by 50%.
• This correlated with reduced cell proliferation, decreased nuclear Sp1 protein, and 50-60% lower Sp1-mediated transactivation.
• EGF and okadaic acid (phosphatase inhibitor) increased Sp1 phosphorylation and synergistically decreased Sp1 protein levels.
• Cycloheximide and leupeptin (cysteine protease inhibitor) blocked EGF-induced Sp1 loss, indicating EGF promotes Sp1 proteolysis.
• Sp1 contains a PEST-like sequence, suggesting susceptibility to regulated degradation.

Conclusions:

• Sustained EGF stimulation triggers a cascade involving phosphorylation events that enhance Sp1 proteolysis.
• This leads to reduced nuclear Sp1 levels and consequently, decreased cellular proliferation in GH4 cells.