Methods for studying signal-dependent regulation of translation factor activity.

The translational machinery of mammalian cells is regulated through the phosphorylation of a number of its components, especially translation factor proteins. These include factors involved in the initiation and elongation stages of translation, and proteins that modify their activity. Examples include eukaryotic initiation factor (eIF) 4E, eukaryotic elongation factor (eEF) 2, and eIF4E-binding protein 1 (4E-BP1). Their phosphorylation is mediated by protein kinases that, in turn, are regulated by specific intracellular signaling pathways. These pathways include those mediated via the mammalian target of rapamycin (mTOR), the ERK and p38 MAP kinase pathways, and protein kinase B (Akt). These pathways are activated by hormones (e.g., insulin), growth factors, mitogens, and other extracellular stimuli. In some cases, amino acids also modulate the pathway (e.g., mTOR). Procedures are described for determining the states of phosphorylation and/or activity of several translation factors, and of kinases that phosphorylate them. We also outline procedures for assessing the states of activation of relevant signaling pathways. In addition, we provide guidelines on using small molecule inhibitors to assess the involvement of specific signaling pathways in controlling translation factors and protein synthesis.