Inhibition of protein synthesis by antagonists of calmodulin in Ehrlich ascites tumor cells

Abstract

Several recent publications indicate that Ca2+ is required for protein synthesis in mammalian cells, including the Ehrlich ascites tumor cell. The present communication examines whether the effects of Ca2 + might be mediated through calmodulin or a related protein. Four calmodulin antagonists belonging to different chemical categories were used to provide evidence of calmodulin involvement. Three of the antagonists inhibited protein synthesis in intact cells; 50% inhibitory concentrations were 10 pM calmidazolium, 12 pM N-(6-aminohexyl)-5-chloro-lnaphthalenesulfonamide (W,) and 17.5 pM trifluoperazine (TFP). Initiation was preferentially inhibited as indicated by an increase in the 80s monomers accompanied by a significant disaggregation of polyribosomes. All the antagonists also inhibited protein synthesis initiation in the cell-free protein-synthesizing system; 50% inhibitory concentrations for compound 48/80, calmidazolium, TFP, and W7 were 10 pM, 125 pM, 300 pM and 500 pM, respectively. A weak analogue of W7 inhibited only 20% at 1000 pM. Inhibition in the cell-free system was reversed by the addition of exogenous calmodulin in all four cases. The levels of 43s complexes were significantly elevated with all four antagonists, indicating a block in the utilization of 43s complexes.The similarity of the effects of four distinct classes of antagonists and their ready reversal by exogenous calmodulin leads us to suggest that there may be a role for calmodulin or a very similar calcium-binding protein in protein synthesis.Calmodulin (CaM), the ubiquitous calcium-binding regulatory protein, is considered to play a pivotal role in eukaryotic cells [l]. It is a member of a family of calcium-binding proteins with similar physical, chemical and sometimes functional properties [2]. CaM has four binding sites for calcium ions and, upon calcium binding, undergoes a large conformational change, exposing a hydrophobic region. Through this hydrophobic region CaM interacts with other proteins and exerts its regulatory effects, conferring calcium sensitivity on a multitude of enzyme systems and cell functions [3].Calmodulin levels increase during periods of cell growth and differentiation [4 -61. Small (2 -3-fold) increases in calmodulin content of transformed cells have been reported [6 -81. This and other indirect evidence has stimulated interest in the possibility that CaM may be involved in regulating cell growth and transformation, but this attractive hypothesis is still speculative.Brostrom and co-workers have shown that depletion of cellular calcium causes a marked inhibition of protein synthesis in a wide variety of cell types [9]. Our earlier data demonstrate an inhibition of protein synthesis in Ehrlich cellsCorrespondence to E. C. Henshaw, University of Rochester Medical Center, 601 Elmwood Avenue, Box 704, Rochester, New York 14642, USA Abbreviations. CaM, calmodulin; W,, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; W5, N-(6-aminohexyl)-naphthalenesulfonamide; TFP, trifluoperazine; Me2S0, dimethyl sulfoxide;...

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