The synthesis of new protein is a highly regulated process that allows rapid cellular responses to diverse stimuli in the absence of transcription. Initiation of protein synthesis begins after the separation of the ribosome into its 40S and 60S subunits. Different eukaryotic initiation factors (eIFs) catalyze the assembly of a functional ribosomal complex including the 40S subunit, mRNA and tRNA, and finally the 60S subunit before the first peptide bond is formed. eIF4F and p70 S6 kinase play critical roles in translational regulation. eIF4F is a complex whose functions include the recognition of the mRNA 5' cap structure (eIF4E), delivery of an RNA helicase to the 5' region (eIF4A), bridging of the mRNA and the ribosome (eIF4G), and circularization of the mRNA via interaction between eIF4G and the poly(A) binding protein (PABP). Several stimuli, including growth factors and cytokines, regulate the eIF4 complex and p70 S6 kinase by initiating a phosphorylation cascade involving the sequential activation of PI3K, PDK1/2, Akt/PKB and FRAP/mTOR kinase.
p70 S6 kinase phosphorylates the 40S subunit ribosomal protein S6 and is involved in translational control of 5' oligopyrimidine tract mRNAs (1). The activity of p70 S6 kinase is controlled by its phosphorylation at multiple sites, with the threonine 389 position shown to correlate most clearly with activity in vivo (2). Phosphorylation of ribosomal protein S6 by p70 S6 kinase stimulates the translation of mRNAs with a 5' oligopyrimidine tract that typically encodes components of the protein synthesis apparatus.
Akt activation, which occurs via phosphorylation within the C-terminus at Ser473 and within the activation loop at Thr308 (3), functions in a wortmannin-sensitive pathway involving PI3 kinase (4–6). LY294002 (PI3 Kinase Inhibitor) was also shown to act in vivo as a highly selective inhibitor of PI3 kinase but not of other lipid and protein kinases such as PI4 kinase, PKC, MAP kinase or c-Src (7). Rapamycin (FRAP/mTOR Inhibitor) is also a valuable tool used in studying pathways regulating translation. Rapamycin treatment of cells leads to the dephosphorylation and inactivation of p70 S6 kinase. It also leads to the dephosphorylation of 4EBP1, promoting its binding to and inactivation of eIF4E (8–10). 4E-BP1 functions in the PI3/Akt pathway and is phosphorylated by FRAP/mTOR at Thr37 and Thr46 (11–13). In its nonphosphorylated state 4E-BP1 binds eIF4E, inhibiting cap-dependent translation. Hyperphosphorylation of 4E-BP1 disrupts this binding, activating cap-dependent translation (9).
Phosphorylation at other sites, including Ser65 and Thr70, by an as yet unknown kinase is required for full inactivation of 4E-BP1. Another mechanism for eIF4E regulation is direct phosphorylation by Mnk1/2. Erk and p38 MAPK can both activate Mnk1/2 to phosphorylate eIF4E at Ser209. Both processes contribute to the association of eIF4E and eIF4G to form the active eIF4F complex, a necessary component of the 48S initiation complex.
The Translational Control Sampler Kit provides a fast and economical means to evaluate the activity of pathways regulating translation, including phosphorylated Akt, elF4E, 4E-BP1 and p70 S6 kinase. The kit includes enough primary and secondary antibodies to perform four mini-blot experiments, as well as specific inhibitors of PI3 kinase and FRAP/mTOR.
仕様
Size：6x20ul
Grade：Affinity Purified
Purity：Purified by Protein A and peptide affinity chromatography.
Form：Supplied as a liquid in 10mM sodium HEPES, pH 7.5, 150mM sodium chloride, 0.1mg/ml BSA, 50% glycerol.
