Physiological stresses inhibit guanine-nucleotide-exchange factor in Ehrlich cells

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Phosphorylation of eukaryotic initiation factor 2 alpha (eIF-2 alpha) increases under physiological stress, inhibiting protein synthesis in Ehrlich ascites tumor cells.
• Phosphorylated eIF-2 [eIF-2(alpha P)] is a known inhibitor of guanine nucleotide exchange factor (GEF), suggesting its role in stress-induced inhibition.

Purpose of the Study:

• To investigate the role of eIF-2 alpha phosphorylation in inhibiting GEF activity under various stress conditions in Ehrlich ascites tumor cells.
• To determine if GEF activity is regulated solely by eIF-2 alpha phosphorylation or by other mechanisms.

Main Methods:

• Assayed GEF activity in cell extracts from Ehrlich cells subjected to heat shock, serum deprivation, and glutamine deprivation.
• Measured GEF activity by its ability to enhance the release of [alpha-32P]GDP from purified eIF-2.
• Investigated the effect of increasing eIF-2.GDP substrate concentration on GEF activity inhibition.

Main Results:

• GEF activity was reduced in heat-shocked and serum-deprived cells, correlating with increased eIF-2 alpha phosphorylation.
• Inhibition of GEF activity in these cells was fully reversed by adding more eIF-2.GDP, confirming eIF-2 alpha phosphorylation as the sole inhibitory mechanism.
• In glutamine-deprived cells, GEF activity was partially inhibited and could not be fully reversed by eIF-2.GDP, suggesting additional regulatory pathways.

Conclusions:

• eIF-2 alpha phosphorylation is the primary mechanism inhibiting GEF activity and protein synthesis under heat shock and serum deprivation in Ehrlich cells.
• Ehrlich cells possess a higher GEF to eIF-2 ratio than reticulocytes, allowing for continued protein synthesis despite partial eIF-2 phosphorylation.
• Glutamine deprivation may involve additional regulatory mechanisms for GEF activity beyond eIF-2 alpha phosphorylation.