Regulation of an eukaryotic initiation factor-2 associated 67 kDa glycoprotein ( p67) during heat shock in animal cells

Eukaryotic initiation factor-2 (eIF-2) associated polypeptide (p$\sp{67}$) plays an important role in the regulation of protein synthesis initiation in the mammalian cells. Under certain physiological conditions, phosphorylation of the $\alpha$-subunit of eIF-2 by kinases leads to eIF-2 inactivation and thereby protein synthesis inhibition. p$\sp{67}$ can protect the eIF-2$\alpha$ from inhibitory phosphorylation and therefore promotes protein synthesis even in the presence of eIF-2 kinases. p$\sp{67}$ level in the cells is regulated by two different mechanisms, (i) by the activation of a p$\sp{67}$-deglycosylase. Heme-deficient reticulocyte and vaccinia viral infected animal cells use this mechanism. A p$\sp{67}$-deglycosylase has been purified from heme deficient reticulocyte lysate. (ii) at the mRNA level. This mechanism is used in serum starvation and heat shock. Regulation of p$\sp{67}$ at the transcriptional level in response to heat shock was studied in rat hepatoma (KRC-7) cells. Cloning and sequence analysis of the 5$\sp\prime$ upstream region of p$\sp{67}$ genomic DNA revealed the presence of heat shock regulatory element. Transcriptional regulation of p$\sp{67}$ in response to heat shock was studied by deleting HSE from p$\sp{67}$ promoter. Deletion of HSE caused significant decrease in the promoter activity upon heat shock treatment. Heat shock resulted in a time dependent prolonged elevation upon heat shock treatment. Heat shock resulted in a time dependent prolonged elevation in the level of p$\sp{67}$ mRNA, which was accompanied by increased translation of p$\sp{67}$ protein. It was observed that a p$\sp{67}$-deglycosylase was activated during 15 min of heat shock and p$\sp{67}$ mostly existed in deglycosylated form during this phase. Loss of glycosyl residues obliterated p$\sp{67}$ activity and it no longer protected eIF-2$\alpha$ from inhibitory phosphorylation. However the level of eIF-2$\alpha$ phosphorylation decreased after 30 min of heat shock. During this second phase, newly synthesized p$\sp{67}$ protein was observed and was found to be glycosylated. Decrease in the level of eIF-2$\alpha$ phosphorylation was paralleled by an induction of p$\sp{67}$ in the glycosylated form