The tumour promoter okadaic acid inhibits reticulocyte-lysate protein synthesis by increasing the net phosphorylation of elongation factor 2.

Okadaic acid, a tumour promoter which potently inhibits protein phosphatases, inhibited translation in the reticulocyte-lysate cell-free system. Inhibition was dose-dependent, with half-maximal effects occurring at 20-40 nM-okadaic acid. Inhibition of translation by okadaic acid resulted in the accumulation of polyribosomes, indicating that it was due to a decrease in the rate of elongation relative to initiation. Okadaic acid (at concentrations which inhibited translation) caused increased phosphorylation of a number of proteins in the lysate. Prominent among these was a protein of Mr 100,000, which has previously been identified as elongation factor 2 (EF-2). EF-2 is a specific substrate for a Ca2+/calmodulin-dependent protein kinase, which phosphorylates EF-2 on threonine residues. The Mr-100,000 band was phosphorylated exclusively on threonine residues, and its degree of 32P labelling was decreased by the Ca2+ chelator EGTA and by the calmodulin antagonist trifluoperazine. These agents attenuated the effects of okadaic acid on EF-2 phosphorylation and translation. When ranges of concentrations of each agent were tested, their effects on EF-2 labelling correlated well with their ability to reverse the okadaic acid-induced inhibition of translation. These findings demonstrate that increased phosphorylation of EF-2 results in an impairment of peptide-chain elongation when natural mRNA is used. The possible physiological role of EF-2 phosphorylation in the control of translation is discussed.