Phosphorylation of proteins, catalyzed by kinases, is critical to cell functions involved with signal transduction. This process involves a change in the phosphorylation status of downstream proteins by modification of specific serine/threonine or tyrosine residues. Inappropriate or deregulated phosphorylation is often associated with diseases, such as diabetes, cancer, heart disease, and neurological and autoimmune diseases. A clear understanding of phosphorylation associated with a specific signaling pathway is the key to understanding pathway activation, related diseases and ultimate treatment. Many end proteins of signal transduction are transcription factors (TFs). TFs usually contain two functional domains, a DNA binding domain (DBD), for interaction with promoters of genes, and a trans-activation domain (TAD), for modulation of the activity of transcriptional machinery. The activity of TADs is often regulated by kinases, with different TADs being modified by specific kinases. Therefore, these domains become valuable targets for the study of kinases, phosphorylation, signal transduction, and transcriptional activation.
Cells will undergo genotypic changes resulting in reduced responsiveness over time in normal cell culture conditions. Genetic instability is a biological phenomenon that occurs in all stably transfected cells. Therefore, it is critical to prepare an adequate number of frozen stocks at early passages.