Anisomycin and Rapamycin Define an Area Upstream of p70/85^S6k Containing a Bifurcation to Histone H3-HMG-Like Protein Phosphorylation and c-fos-c-jun Induction

Abstract

Anisomycin, a translational inhibitor, synergizes with growth factors and phorbol esters to superinduce c-fos and c-jun by a number mechanisms, one of which is its ability to act as a potent signalling agonist, producing strong, prolonged activation of the same nuclear responses as epidermal growth factor or tetradecanoyl phorbol acetate. These responses include the phosphorylation of pp33, which exists in complexed and chromatin-associated forms, and of histone H3 and an HMG-like protein. By peptide mapping and microsequencing, we show here that pp33 is the phosphoprotein S6, present in ribosomes and in preribosomes in the nucleolus. Ablation of epidermal growth factor-, tetradecanoyl phorbol acetate-, or anisomycin-stimulated S6 phosphorylation by using the p70/85S6k inhibitor rapamycin has no effect on histone H3 and HMG-like protein phosphorylation or on the induction and superinduction of c-fos and c-jun. Further, [35S]methionine-labelling and immunoprecipitation studies show that the ablation of S6 phosphorylation has no discernible effect on translation in general or translation of newly induced c-fos transcripts. Finally, we show that anisomycin augments and prolongs S6 phosphorylation not by blocking S6 phosphatases but by sustained activation of p70/85S6k. These results suggest the possible use of anisomycin and rapamycin to define upstream and downstream boundaries of an area of signalling above p70/85S6k which contains a bifurcation that produces histone H3-HMG-like protein phosphorylation and c-fos-c-jun induction in the nucleus.

Synapse integrity and function: Dependence on protein synthesis and identification of potential failure points

“…In this study we used protein synthesis inhibitors to block the supply of new proteins in neurons. Translational inhibitors, however, have been reported to have widespread effects on neuronal function, some of which might be unrelated to their capacity to suppress protein synthesis: Superinduction of immediate early genes ( Mahadevan and Edwards, 1991 ; Hazzalin et al, 1998 ; Radulovic and Tronson, 2008 ; Santos et al, 2019 ); increased synthesis /hyperproduction of specific proteins ( Törocsik and Szeberényi, 2000a ; Radulovic and Tronson, 2008 ; Kenney et al, 2016 ); activation of signaling pathways ( Cano et al, 1994 ; Kardalinou et al, 1994 ; Zinck et al, 1995 ; Iordanov et al, 1997 ; Hazzalin et al, 1998 ; Iordanov and Magun, 1998 ; Törocsik and Szeberényi, 2000a ; Monaghan et al, 2014 ; Tyssowski et al, 2018 ); apoptosis/cytotoxicity (in certain cell types; Törocsik and Szeberényi, 2000b ; Monaghan et al, 2014 ; Chan et al, 2017 ); effects on protein degradation ( Franklin and Johnson, 1998 ; Dai et al, 2013 ; see also Ding et al, 2007 ; Kaang and Choi, 2012 ; Jarome and Helmstetter, 2014 ); and altered axonal transport ( Levy et al, 1990 ). Despite these reports, the similarity of the findings in experiments based on two different inhibitors (CHX, ANI) suggest that these are unlikely to stem primarily from off-target effects of these inhibitors.…”