Abstract
Akt1, also known as protein kinase B (PKB) alpha, is frequently activated in human cancers and has been implicated in many cell processes by phosphorylation of downstream molecules. However, transcriptional regulation of Akt1 has not been documented. Here, we report the isolation and characterization of the human AKT1 promoter and demonstrate transcriptional up-regulation of AKT1 by the Src/Stat3 pathway. Protein and mRNA levels of AKT1 are elevated in cells expressing constitutively active Stat3 as well as in v-Src-transformed NIH3T3 cells. Knockdown of Stat3 reduces AKT1 expression induced by v-Src. Although the 4.2-kb region upstream of the transcription start site of the AKT1 promoter contains five putative Stat3-binding motifs, the promoter failed to be induced by Stat3 and/or Src. Further analysis reveals that major Stat3 response elements are located within exon 1 and intron 1 regions of the AKT1 gene, which is upstream of the AKT1 translation initiation site. In addition, ectopic expression of wild type AKT1 in Stat3(-/-) MEF cells largely rescues serum starvation-induced cell death. These findings indicate that the AKT1 promoter comprises exon 1 and intron 1, in addition to the sequence upstream of transcriptional start site. Our data further show that AKT1 is a direct target gene of Stat3 and contributes to Stat3 anti-apoptotic function.
Highlights
Akt[1], known as protein kinase B (PKB) ␣, is frequently acti- mutated in a number of human malignancies
The activity of Akt is negatively regulated by PTEN, a tumor suppressor gene that is invasion and metastasis in human breast and ovarian cancer cells (25) and induces a malignant phenotype in mouse fibroblasts (20)
Stat[3] Transactivates the AKT1 Promoter—We further examined whether the AKT1 promoter is regulated by Stat[3]
Summary
Cell Culture, Plasmids, Materials, and Transfection—Human epithelial kidney HEK293, MCF10A, MCF7, mouse fibroblast NIH3T3, Srctransformed NIH3T3, and Stat3Ϫ/Ϫ MEF were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. Luciferase Reporter Assay—NIH3T3 or HEK293 cells were cultured in 12-well plates and transiently transfected with AKT1-Luc, Src, and/or Stat[3]. Solubilized chromatin was prepared from a total of 2 ϫ 107 asynchronously growing HEK293 cells that were transfected with wild type Stat[3] and v-Src. The chromatin solution was diluted 10-fold with ChIP dilution buffer (1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl, 16.7 mM Tris-HCl, pH 8.1, 0.01% SDS, protease inhibitors), and precleared with protein-A beads blocked with 2 g of sheared salmon sperm DNA and preimmune serum. Purified DNA was subjected to PCR with primers specific for 13 putative Stat3-binding sites within the AKT1 promoter.
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