Eukaryotic initiation factor 2B: identification of multiple phosphorylation sites in the epsilon-subunit and their functions in vivo

Eukaryotic initiation factor (eIF) 2B is a heteromeric guanine nucleotide e xchange factor that plays an important role in regulating mRNA translation. Here we identify multiple phosphorylation sites in the largest, catalytic, subunit (epsilon) of mammalian eIF2B. These sites are phosphorylated by fo ur different protein kinases. Two conserved sites (Ser712/713) are phosphor ylated by casein kinase 2. They lie at the extreme C-terminus and are requi red for the interaction of eIF2B epsilon with its substrate, eIF2, in vivo and for eIF2B activity in vitro. Glycogen synthase kinase 3 (GSK3) is respo nsible for phosphorylating Ser535. This regulatory phosphorylation event re quires both the fourth site (Ser539) and a distal region, which acts to rec ruit GSK3 to eIF2B epsilon in vivo. The fifth site, which lies outside the catalytic domain of eIF2B epsilon, can be phosphorylated by casein kinase 1 . All five sites are phosphorylated in the eIF2B complex in vivo.