ABSTRACT
Overexpression of epidermal growth factor receptor (EGFR) in certain cancers is well established. There is growing evidence that epidermal growth factor (EGF) activates Akt/protein kinase B (PKB) in a phosphoinositide 3-OH kinase (PI3K)-dependent manner, but it is not yet clear which Akt isoforms are involved in this signal transduction pathway. We investigated the functional regulation of three Akt isoforms, Akt1/PKBalpha, Akt2/PKBbeta, and Akt3/PKBgamma, in esophageal cancer cells where EGFR is frequently overexpressed. Upon EGF simulation, phosphorylation of Akt1 at the Ser-473 residue was remarkably induced. This result was corroborated by in vitro Akt kinase assays using glycogen synthase kinase 3beta as the substrate. PI3K inhibitors, wortmannin or LY294002, significantly blocked the Akt kinase activity induced by EGF. Akt2 activity was evaluated by electrophoretic mobility shift assays. Robust activation of Akt2 by EGF was observed in some cell lines in a PI3K-dependent manner. EGF-induced Akt3 activation was demonstrated by Ser-472 phosphorylation of Akt3 but in a restrictive fashion. In aggregate, EGF-mediated activation of Akt isoforms is overlapping and distinctive. The mechanism by which EGFR recruits the PI3K/Akt pathway was in part differentially regulated at the level of Ras but independent of heterodimerization of EGFR with either ErbB2 or ErbB3 based upon functional dissection of pathways in esophageal cancer cell lines.
Subject(s)
Epidermal Growth Factor/metabolism , Oncogene Proteins/chemistry , Protein Serine-Threonine Kinases/chemistry , Proto-Oncogene Proteins/chemistry , Androstadienes/pharmacology , Blotting, Southern , Blotting, Western , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Line , Chromones/pharmacology , Dimerization , Enzyme Activation , Enzyme Inhibitors/pharmacology , Esophageal Neoplasms/metabolism , Glycogen Synthase Kinase 3 , Glycogen Synthase Kinases , Humans , Ligands , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Precipitin Tests , Protein Isoforms , Proto-Oncogene Proteins c-akt , Receptor, ErbB-2/chemistry , Receptor, ErbB-2/metabolism , Receptor, ErbB-3/chemistry , Receptor, ErbB-3/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Serine/metabolism , Signal Transduction , Time Factors , Transfection , Tumor Cells, Cultured , Tyrosine/metabolism , Wortmannin , ras Proteins/chemistry , ras Proteins/metabolismABSTRACT
Early in infection of Bacillus subtilis by bacteriophage SPO1, the synthesis of most host-specific macromolecules is replaced by the corresponding phage-specific biosyntheses. It is believed that this subversion of the host biosynthetic machinery is accomplished primarily by a cluster of early genes in the SPO1 terminal redundancy. Here we analyze the nucleotide sequence of this 11.5-kb "host-takeover module," which appears to be designed for particularly efficient expression. Promoters, ribosome-binding sites, and codon usage statistics all show characteristics known to be associated with efficient function in B. subtilis. The promoters and ribosome-binding sites have additional conserved features which are not characteristic of their host counterparts and which may be important for competition with host genes for the cellular biosynthetic machinery. The module includes 24 genes, tightly packed into 12 operons driven by the previously identified early promoters PE1 to PE12. The genes are smaller than average, with half of them having fewer than 100 codons. Most of their inferred products show little similarity to known proteins, although zinc finger, trans-membrane, and RNA polymerase-binding domains were identified. Transcription-termination and RNase III cleavage sites were found at appropriate locations.
