Mouse anti-Human S6 (pS235/pS236) monoclonal antibody, clone O8-659 [V450]

Pasteurella multocida toxin (PMT) is a potent mitogen known to activate several signaling pathways via deamidation of a conserved glutamine residue in the alpha subunit of heterotrimeric G-proteins. However, the detailed mechanism behind mitogenic properties of PMT is unknown. Herein, we show that PMT induces protein synthesis, cell migration, and proliferation in serum-starved Swiss 3T3 cells. Concomitantly PMT induces phosphorylation of ribosomal S6 kinase (S6K1) and its substrate, ribosomal S6 protein (rpS6), in quiescent 3T3 cells. The extent of the phosphorylation is time and PMT concentration dependent, and is inhibited by rapamycin and Torin1, the two specific inhibitors of the mammalian target of rapamycin complex 1 (mTORC1). Interestingly, PMT-mediated mTOR signaling activation was observed in MEF WT but not in G alpha(q/11) knock-out cells. These observations are consistent with the data indicating that PMT-induced mTORC1 activation proceeds via the deamidation of G alpha(q/11), which leads to the activation of PLC beta to generate diacylglycerol and inositol trisphosphate, two known activators of the PKC pathway. Exogenously added diacylglycerol or phorbol 12-myristate 13-acetate, known activators of PKC, leads to rpS6 phosphorylation in a rapamycin-dependent manner. Furthermore, PMT-induced rpS6 phosphorylation is inhibited by PKC inhibitor, Go6976. Although PMT induces epidermal growth factor receptor activation, it exerts no effect on PMT-induced rpS6 phosphorylation. Together, our findings reveal for the first time that PMT activates mTORC1 through the G alpha(q/11)/PLC beta/PKC pathway. The fact that PMT-induced protein synthesis and cell migration is partially inhibited by rapamycin indicates that these processes are in part mediated by the mTORC1 pathway.

This study aimed to determine (1) the effect of acute resistance exercise on mechanisms of ribosome biogenesis, and (2) the impact of mammalian target of rapamycin on ribosome biogenesis, and muscle protein synthesis (MPS) and degradation. Female F344BN rats underwent unilateral electrical stimulation of the sciatic nerve to mimic resistance exercise in the tibialis anterior (TA) muscle. TA muscles were collected at intervals over the 36h of exercise recovery (REx); separate groups of animals were administered rapamycin pre-exercise (REx+Rapamycin). Resistance exercise led to a prolonged (6-36h) elevation (30-50%) of MPS that was fully blocked by rapamycin at 6h but only partially at 18h. REx also altered pathways that regulate protein homeostasis and mRNA translation in a manner that was both rapamycin-sensitive (proteasome activity; phosphorylation of S6K1 and rpS6) and rapamycin-insensitive (phosphorylation of eEF2, ERK1/2 and UBF; gene expression of the myostatin target Mighty as well as c-Myc and its targets involved in ribosome biogenesis). The role of c-Myc was tested in vitro using the inhibitor 10058-F4, which, over time, decreased basal RNA and MPS in a dose-dependent manner (correlation of RNA and MPS, r(2)=0.98), even though it had no effect on the acute stimulation of protein synthesis. In conclusion, acute resistance exercise stimulated rapamycin-sensitive and -insensitive mechanisms that regulate translation activity and capacity.