Changes in the phosphorylation of eukaryotic initiation factor 2 alpha, initiation factor 2B activity and translational rates in primary neuronal cultures under different physiological growing conditions.
Phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2) is one of the best known mechanisms regulating protein synthesis in a wide range of eukaryotic cells, from yeast to human. To determine whether this mechanism operates in primary neuronal cells, we have cultured primary neuronal cells for 7 days under two optimal growing conditions, complete medium (containing 15% serum) and serum-free medium, and determined the protein synthesis rate, eukaryotic initiation 2 and 2B (eIF-2B) activities, as well as the level of phosphorylation of eIF-2. Cells cultured in serum-free medium exhibited a lower rate of protein synthesis (75%), concomitant to a decreased eIF-2 activity (71%), and slightly higher eIF-2(alpha P) levels (from 10 to 16% of total eIF-2) with respect to cells cultured in complete media. eIF-2B activity, as measured at saturating eIF-2. GDP concentrations (assay independent on the presence of eIF-2(alpha P)) was similar under the two culture conditions. When neurons cultured in serum-free medium are exposed to complete medium for only 24 h, there is a clear decrease in the phosphorylation of eIF-2 alpha (16-3%). This decrease correlates in time with an increase in the protein synthesis rate (154%), as well as eIF-2 activity (236%). The increased levels of eIF-2(alpha P), a competitive inhibitor of eIF-2B in the guanine-exchange reaction, are responsible for the decreased eIF-2B activity found in the neurons cultured in serum-free medium. Additionally, eIF-2(alpha P) is accountable for the lower effect of exogenous eIF-2B in ternary complex formation from preformed eIF-2. GDP in the serum-free media. These changes in phosphorylation of eIF-2 alpha in normal mammalian cells in response to changes in the extracellular medium are reported here for the first time.
Alcázar
A
A
Departamento de Investigación, Hospital Ramón y Cajal, Madrid, Spain.
Rivera
J
J
Gómez-Calcerrada
M
M
Muñoz
F
F
Salinas
M
M
Fando
J L
JL
D000818:Animals; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D003470:Culture Media; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism*; D051381:Rats
D003470:Culture Media; D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D009419:Nerve Tissue Proteins; D011506:Proteins