Multiple mechanisms control phosphorylation of PHAS-I in five (S/T)P sitesthat govern translational repression

Control of the translational repressor, PHAS-I, was investigated by express ing proteins with Ser/Thr--> Ala mutations in the five (S/T)P phosphorylati on sites. Results of experiments with HEK293 cells reveal at least three le vels of control. At one extreme is nonregulated phosphorylation, exemplifie d by constitutive phosphorylation of Ser82. At an intermediate level, amino acids and insulin stimulate the phosphorylation of Thr36, Thr45, and Thr69 via mTOR-dependent processes that function independently of other sites in PHAS-I. At the third level, the extent of phosphorylation of one site modu lates the phosphorylation of another. This control is represented by Ser64 phosphorylation, which depends on the phosphorylation of all three TP sites . The five sites have different influences on the electrophoretic propertie s of PHAS-I and on the affinity of PHAS-I for eukaryotic initiation factor 4E (eIF4E). Phosphorylation of Thr45 or Ser64 results in the most dramatic decreases in eIF4E binding in vitro. However, each of the sites influences mRNA translation, either directly by modulating the binding affinity of PHA S-I and eIF4E or indirectly by affecting the phosphorylation of other sites .