磷酸化
AKT1型
HEK 293细胞
生物
激酶
细胞生物学
丝氨酸
蛋白激酶B
生物化学
基因
作者
Tarana Siddika,Rong‐Guang Shao,Ilka U. Heinemann,Patrick O’Donoghue
摘要
Abstract Protein kinase B (AKT1) is a serine/threonine kinase that regulates fundamental cellular processes, including cell survival, proliferation, and metabolism. AKT1 activity is controlled by two regulatory phosphorylation sites (Thr308, Ser473) that stimulate a downstream signaling cascade through phosphorylation of many target proteins. At either or both regulatory sites, hyperphosphorylation is associated with poor survival outcomes in many human cancers. Our previous biochemical and chemoproteomic studies showed that the phosphorylated forms of AKT1 have differential selectivity toward peptide substrates. Here, we investigated AKT1‐dependent activity in human cells, using a cell‐penetrating peptide (transactivator of transcription, TAT) to deliver inactive AKT1 or active phospho‐variants to cells. We used enzyme engineering and genetic code expansion relying on a phosphoseryl‐transfer RNA (tRNA) synthetase (SepRS) and tRNA Sep pair to produce TAT‐tagged AKT1 with programmed phosphorylation at one or both key regulatory sites. We found that all TAT‐tagged AKT1 variants were efficiently delivered into human embryonic kidney (HEK 293T) cells and that only the phosphorylated AKT1 (pAKT1) variants stimulated downstream signaling. All TAT‐pAKT1 variants induced glycogen synthase kinase (GSK)‐3α phosphorylation, as well as phosphorylation of ribosomal protein S6 at Ser240/244, demonstrating stimulation of downstream AKT1 signaling. Fascinatingly, only the AKT1 variants phosphorylated at S473 (TAT‐pAKT1 S473 or TAT‐pAKT1 T308,S473 ) were able to increase phospho‐GSK‐3β levels. Although each TAT‐pAKT1 variant significantly stimulated cell proliferation, cells transduced with TAT‐pAKT1 T308 grew significantly faster than with the other pAKT1 variants. The data demonstrate differential activity of the AKT1 phospho‐forms in modulating downstream signaling and proliferation in human cells.
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