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

Previously, we have shown that phosphorylation of the eukaryotic initiation factor eIF-2 alpha increases under several physiological stresses in which protein synthesis is inhibited in Ehrlich ascites tumor cells. As phosphorylated eIF-2 [eIF-2(alpha P)] is a potent inhibitor of guanine nucleotide exchange factor (GEF), it seemed likely that it was responsible for the inhibition. We have assayed GEF activity levels in extracts prepared from Ehrlich cells exposed to three such stresses, namely heat shock, serum deprivation and glutamine deprivation. Activity was estimated by the ability of GEF to enhance the release of [alpha-32P]GDP from purified eIF-2 [a modification of the reticulocyte lysate assay of Matts, R. L. & London, I. M. (1984) J. Biol. Chem. 259, 6708]. GEF activity was reduced from control values in extracts of heat-shocked cells and serum-deprived cells, concomitant with increased eIF-2 alpha phosphorylation. Inhibition of GEF activity in heat-shocked and serum-deprived cells was reversed to control levels by increasing the concentration of purified eIF-2.GDP added as substrate in the GEF assay. Since we have shown elsewhere that eIF-2(alpha P).GDP inhibits GEF by competition with eIF-2.GDP, the complete reversal of inhibition of GEF activity in heat-shocked and serum-deprived cells indicates that inhibition is due solely to phosphorylation of eIF-2 alpha. In glutamine-deprived cells phosphorylation of eIF-2 alpha was increased modestly and GEF activity was reduced but GEF activity could not be fully reversed by addition of eIF-2.GDP, suggesting that GEF may also be regulated in other ways. There are greater amounts of GEF relative to eIF-2 in Ehrlich cells (approximately 50%) compared with rabbit reticulocytes (approximately 20%). This explains the efficient rates of protein synthesis in control Ehrlich cells even though they have 30% of their eIF-2 phosphorylated which is enough to inhibit GEF and initiation in reticulocytes completely but only enough to trap approximately 60% of the GEF in Ehrlich cells.

The effect of Mg2+ and guanine nucleotide exchange factor on the binding of guanine nucleotides to eukaryotic initiation factor 2.

A major site of regulation of polypeptide chain initiation is the binding of Met-tRNA to 40 S ribosomal subunits which is mediated by eukaryotic initiation factor 2 (eIF-2). The formation of ternary complex, eIF-2.GTP.Met-tRNA, is potently inhibited by GDP. Measurement of the parameters for guanine nucleotide binding to eIF-2 is critical to understanding the control of protein synthesis by fluctuations in cellular energy levels. We have compared the dissociation constants (Kd) of eIF-2.GDP and eIF-2.GTP and find that GDP has a 400-fold higher affinity for GDP than GTP. The Kd for GDP is almost an order of magnitude less than has been reported previously. The difference between the Kd values for the two nucleotides is the result of a faster rate constant for GTP release, the rate constants for binding being approximately equal. This combination of rate constants and low levels of contaminating GDP in preparations of GTP can explain the apparently unstable nature of eIF-2.GTP observed by others. Mg2+ stabilizes binary complexes slowing the rates of release of nucleotide from both eIF-2.GDP and eIF-2.GTP. The competition between GTP and GDP for binding to eIF-2.guanine nucleotide exchange factor complex has been measured. A 10-fold higher GTP concentration than GDP is required to reduce [32P] GDP binding to eIF-2.guanine nucleotide exchange factor complex by 50%. The relevance of this competition to the regulation of protein synthesis by energy levels is discussed.

The catalytic mechanism of guanine nucleotide exchange factor action and competitive inhibition by phosphorylated eukaryotic initiation factor 2.

Guanine nucleotide exchange factor (GEF) is a multisubunit protein involved in the initiation of translation. Although numerous models have been proposed for its mechanism of action, none have been definitive. An assay dependent on GEF activity was developed using highly purified eukaryotic initiation factor 2 (eIF-2) and GEF from Ehrlich cells. GEF was considered in terms of an enzyme whose catalytic function was the exchange of eIF-2-bound [alpha-32P]GDP for unlabeled nucleotide. The turnover number of GEF at 37 degrees C, calculated on the basis of enzyme kinetic methods is 0.027 s, which is consistent with in vivo rates of protein synthesis. Moreover, kinetic data support an enzyme-substituted mechanism as the mode of GEF function. This mechanism proposes the existence of a GEF.eIF-2.GDP complex and excludes the possibility of two guanine nucleotide binding sites on eIF-2. An analogous mechanism has been recently reported for elongation factor Ts, suggesting the importance of this mechanism to protein synthesis. The mechanism of inhibition of GEF function by eIF-2 alpha phosphorylation has also been investigated. It has been generally assumed that the mechanism by which eIF-2(P) traps GEF is an excessively stable complex, from which GEF is released very slowly. Data presented here, however, reveal that eIF-2(P).GDP is a competitive inhibitor of GEF (rather than an irreversible inhibitor) competing with eIF-2.GDP for binding to GEF. Even though the eIF-2(P).GDP.GEF complex dissociates too rapidly to measure, GEF is trapped because it has at least 150-fold greater affinity for eIF-2(P).GDP than for eIF-2.GDP. The implications of competitive inhibition with respect to the mechanism of reversal of inhibition by an eIF-2(P) phosphatase are discussed.

Phosphorylation of eukaryotic initiation factor 2 during physiological stresses which affect protein synthesis.

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2) is a major mechanism regulating protein synthesis in rabbit reticulocytes. To determine whether phosphorylation of eIF-2 alpha is a likely regulatory mechanism in the Ehrlich cell, we have measured the percent of cellular eIF-2 alpha which is phosphorylated in cells exposed to heat shock, 2-deoxyglucose, or amino acid deprivation, conditions which rapidly decrease the concentration of 40 S initiation complexes and inhibit protein synthesis. eIF-2 alpha and eIf-2 alpha (P) were separated by isoelectric focusing and were detected by immunoblotting with a monoclonal antibody we developed for this purpose. Under the above three inhibitory conditions, phosphorylation of eIF-2 alpha increased rapidly, and this increase correlated in time with the rapid inhibition of protein synthesis. In heat-shocked cells which were returned to 37 degrees C, both phosphorylation and protein synthesis remained unchanged for 10 min and then returned toward control values slowly and in parallel. The close temporal correspondence between changes in protein synthesis and phosphorylation supports an important regulatory role for phosphorylation in protein synthesis. An increase of 25-35 percentage points, to 50-60% phosphorylation from control levels of 20-30% phosphorylation, correlated with an 80-100% inhibition of protein synthesis. This steep curve of inhibition is consistent with a mechanism in which eIF-2 alpha (P) saturates and inhibits the guanine-nucleotide exchange factor.