Anti-4E Binding Protein 1 (Protein Synthesis Initiation Factor 4E Binding Protein, eIF-4EBP1, PHAS-1

PHAS-I, also known as eIF4E-BP1 and PHAS-II,-III (eIF4E-BP2, 3), are members of a family of proteins that regulate eukaryotic translation initiation which is mediated by the cap structure (m7GpppN, where N=any nucleotide) present at the 5í end of all cellular mRNAs, except organellar (1). The m7 cap is essential for the translation of most mRNA because it directs the translation machinery of the 5í end of the mRNA via its interaction with the cap binding protein, the translation initiation factor 4E(eIF4E) (2). eIF4E plays an principal role in determining global translation rates because its interaction with the cap facilitates the binding of the ribosome to the mRNA. Consistent with this role, eIF4E is required for cell cycle progression, exhibits anti-apoptotic activity and when overexpressed transforms cells (2). Interaction with PHAS proteins prevents incorporation of eIF4E into an active translation initiation complex and inhibits cap-dependent translation. However, this inhibitory effect is alleviated following phosphorylation of the PHAS proteins by a P13K-dependent pathway, involving signaling by the anti-apoptotic kinase Akt/PKB, as well as FRAP/mTOR (2). Rat PHAS-I has 117 amino acids with a apparent molecular weight of 22kD and is 93% identical to eIF-4E-BPI cloned from human placenta (3, 4). PHAS-I and ñII were found to have overlapping but different patterns of expression in tissues. PHAS-I is expressed in a wide variety of cell types with the highest being in two of the most insulin-responsive tissues, adipocytes and skeletal muscle (3). Both PHAS proteins are phosphorylated in response to insulin or growth factors such as EGF, PDGF and IGF-1. Increasing cAMP in cells promotes dephosphorylation of both PHAS-I and PHAS-II but that regulation of the two protein differ because PHAS-II, unlike PHAS-I is readily phosphorylated by PKA (5). The PHAS-I initiation factor has 2-8 phosphorylation sites and is multiply phosphorylated by insulin-stimulated protein kinase(s) resulting in 8-10 phosphorylated isoforms in exponentially growing cells. Changes occur in the expression of these isoforms in response to stresses such as heat shock, and this may contribute to translation repression (6). Applications: Suitable for use in Western Blot and Immunohistochemistry. Other applications not tested. Recommended Dilution: Western Blot: 1:500-1:1000, Immunohistochemistry: 1:50-1:100 , Optimal dilutions to be determined by the researcher. Storage and Stability: For long-term storage, aliquot and store at -20°C. Aliquots are stable for at least 12 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.