Inactivation of eIF2B and Phosphorylation of PHAS-I in Heat-shocked Rat Hepatoma Cells

Abstract

Various factors are involved in the heat shock-induced inhibition of protein synthesis. Changes upon heat shock in phosphorylation, leading to inactivation, of eukaryotic initiation factors (eIFs) eIF2 and eIF4E have been shown for several cell types. However, in mammalian cells these changes occur at temperatures of 43°C or higher while protein synthesis is already affected at milder heat shock temperatures. In searching for the cause for the inhibition of protein synthesis, the regulation of eIF2 and eIF4E by additional factors was analyzed. In this respect, the activity of eIF2B was measured during and after heat shock. A very clear correlation was found between the activity of this guanine exchange factor and the levels of protein synthesis, also at mild heat shock conditions. Changes in the phosphorylation of eIF4E and of the eIF4E-binding protein PHAS-I were also analyzed. Surprisingly, in H35 cells as well as in some other cell lines, PHAS-I phosphorylation was increased by heat shock, whereas in others it was decreased. Therefore, decreasing the eIF4E availability under stressful conditions does not seem to be a general mechanism to inhibit protein synthesis by heat shock. Regulation of eIF2B activity appears to be the main mechanism to control translation initiation after heat shock at mild temperatures.Incubating mammalian cells at elevated temperatures inhibits the translation of mRNAs (1-4). The translational lesion in heat-shocked cells principally occurs at the initiation step of translation (1,(5)(6)(7) for review, see Ref. 8). In the initiation process, the 7-methyl guanosine cap at the 5Ј end of the mRNA is bound by a complex of eukaryotic initiation factors (eIFs), 1 including the cap-binding protein eIF4E and the RNA helicase eIF4A. Unwinding of the RNA enables recognition of the AUG initiation codon by the 43 S ribosomal complex and delivery of initiator methionyl tRNA. Then the large ribosomal subunit joins the complex, and peptide synthesis begins (8).Both the concentration and activity of protein synthesis initiation factors and the primary sequence of the 5Ј-untranslated region of an mRNA can affect the rates of eukaryotic translation initiation. With respect to the heat shock-induced modifications of eukaryotic protein synthesis initiation factors, it is of interest that the activity of many initiation factors can be regulated by phosphorylation. It has been shown that changes in the phosphorylation state of various of these eukaryotic initiation factors, such as eIF2, eIF2B, eIF4B, and eIF4E, could coincide with changes in protein synthesis (9, 10).The proteins eIF2 and eIF2B act to bring Met-tRNA to the 40 S ribosomal subunit: eIF2 by binding to Met-tRNA and GTP, and eIF2B by replacing GDP on eIF2 for GTP. Previous studies from several laboratories have focused on heat shock-induced phosphorylation of eIF2␣ (11-13). However, it was found that less severe heat shocks that inhibit protein synthesis by more than 70% do not elicit this phosphorylation. Duncan and Hershey (13) suggested tha...

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