Tyrosine phosphorylation is a highly regulated post-translational modification required for inter- or intra-cellular communication. During this event, the protein tyrosine kinases (PTKs) function as catalyzing enzymes to transfer the terminal phosphate of ATP to tyrosine residues of substrate proteins. PTKs are a large and diverse family of proteins that are found only in Metazoans. Of the identified PTKs in the human genome, 58 are receptor tyrosine kinases (RTKs) distributed into 20 subfamilies, and 32 are non-receptor types. The receptor tyrosine kinases (RTKs) family, characterized by a transmembrane domain and an intrinsic intracellular tyrosine kinase motif, includes epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), Met (hepatocyte growth factor receptor), insulin receptor (IR), and many others. RTKs have been shown to be involved in diverse cell signaling pathways and play key roles in many biological processes including embryogenesis, cellular proliferation, differentiation, migration and apoptosis. While RTK tyrosine kinase activity is tightly controlled in normal cells, unregulated RTK activity through either mutational activation or over-expression is implicated in the development of many diseases like .
Signal transduction pathways are initiated when activated EGFR directly or indirectly recruits various signaling proteins, like She, Grb2, PLC- γ, . Due to the functional diversity of signaling proteins, the activation of these signaling proteins may lead to the assembly of different multicomponent signaling complexes and subsequent activation of multiple signaling pathways, including Ras pathway, PLC- γ 1 pathway and PI3K pathway. As these pathways are linked together directly or indirectly, activation of EGFR actually stimulates a signaling network.
Ras signaling pathway The Ras/Raf/MEK/MAPK pathway mediates numerous cellular functions in different tissues and cell types such as cell proliferation, differentiation, transformation, and survival. EGF-stimulated Ras activation is mediated by the adaptor protein Grb2, which can associate with activated EGFR directly via Y1068 and y1086 sites, or indirectly through tyrosine phosphorylated She. Through binding Grb2, the protein Sos, a guanine nucleotide exchange factor (GEF) for Ras, relocates to EGFR at the plasma membrane and activates membrane-associated Ras, which in turn activates subsequent Raf-MEK-MAPK signaling cascade. Finally, MAPK (Erk) translocates to the nucleus and activates several transcriptional factors such as c-Fos and c-Jun, driving cell proliferation and other processes.
PLC- γ 1 and PI3K signaling pathways Phospholipase C- γ 1 (PLC- γ 1) and phosphatidylinositol-3-kinase (PI3K) pathways are two important EGFR signaling pathways implicated in phospholipid metabolism. Both pathways use PtdIns(4,5)P2 (PIP2) as a substrate, and are involved in cell mitogenesis and motility. Through its SH2 domain, PLC- γ 1 binds directly to phosphorylated EGFR via Y992 and Y1173 sites. Following activation by EGFR, PLC-γ l hydrolyzes PIP2 to yield the important second messengers inositol 1, 4, 5-triphosphate (IP3) and diacylglycerol (DAG), which mediates intracellular calcium release and activates protein kinase C (PKC), respectively. Thus, through PLC-γ l, EGFR can affect Ca2+-dependent induction of NF-κB, and PKCdependent induction of MAPK. Activated EGFR binds to and activates PI3K through its interaction with the p85 subunit of P13K. Activated P13K then catalyses the phosphorylation of PIP2 to produce second messenger PtdIns(3,4,5)P3 (PIP3), which can activate several downstream targets, such as serine/threonine kinase Akt (Protein kinase B, PKB). Akt is the major mediator of the anti-apoptotic effects of the PI3K pathway. Activated Akt promotes cell survival by phosphorylating and inactivating forkhead family of transcription factors, which by themselves induce the expression of proapoptotic factors such as Fas ligand. Akt can also phosphorylate proapoptotic factor BAD, a BCL-2 family member, and suppress BAD-mediated cellular apoptosis.
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