Mothe-Satney I, Yang D, Fadden P, Haystead T A, Lawrence J C
Departments of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Mol Cell Biol. 2000 May;20(10):3558-67. doi: 10.1128/MCB.20.10.3558-3567.2000.
Control of the translational repressor, PHAS-I, was investigated by expressing proteins with Ser/Thr --> Ala mutations in the five (S/T)P phosphorylation sites. Results of experiments with HEK293 cells reveal at least three levels of control. At one extreme is nonregulated phosphorylation, exemplified 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 modulates 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 properties 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 PHAS-I and eIF4E or indirectly by affecting the phosphorylation of other sites.
通过表达在五个（S/T）P磷酸化位点具有丝氨酸/苏氨酸到丙氨酸突变的蛋白质，对翻译阻遏物PHAS-I的调控进行了研究。用HEK293细胞进行的实验结果揭示了至少三个调控水平。在一个极端是无调控的磷酸化，以Ser82的组成型磷酸化为例。在中间水平，氨基酸和胰岛素通过mTOR依赖性过程刺激Thr36、Thr45和Thr69的磷酸化，这些过程独立于PHAS-I中的其他位点起作用。在第三个水平，一个位点的磷酸化程度调节另一个位点的磷酸化。这种调控以Ser64磷酸化为代表，它依赖于所有三个TP位点的磷酸化。这五个位点对PHAS-I的电泳性质以及PHAS-I与真核起始因子4E（eIF4E）的亲和力有不同影响。Thr45或Ser64的磷酸化导致体外eIF4E结合最显著的降低。然而，每个位点都影响mRNA翻译，要么直接通过调节PHAS-I和eIF4E的结合亲和力，要么间接通过影响其他位点的磷酸化。
