Ras Signaling Pathway for Analysis of Protein-Protein Interactions in Yeast and Mammalian Cells
The mitogen-activated protein kinase (MAPK) signaling pathway is initiated by diverse stimuli that occur at the cell membrane and result in a change in pattern of gene expression in the nucleus. Although multiple proteins are involved in the transmission process through a complex cascade of events, the signal is rapidly and efficiently transmitted (1). Protein-protein interaction plays a major role in the transmission process itself as well as in the maintenance of the signaling molecules in an inactive dormant state within the cell. A large variety of mechanisms are involved in the translation of the signal into molecular events. One of the mechanisms involved is the generation of increased local concentration of reactant within a large compartment such as the cytoplasm. A case in point is the plasma membrane. Upon growth factor stimulation, e.g., the inner leaflet of the plasma membrane serves as a subcellular compartment for the recruitment of enzymes in close proximity to their substrates, thereby overcoming the thermodynamic barrier and permitting the enzymatic reaction to occur. The plasma membrane compartment can be subdivided into multiple microenvironments as well (2). Protein recruitment per se is possibly accompanied by phosphorylation and a transient conformational change that results in potentiation of the corresponding enzymatic activity. Autophosphorylation of the cytoplasmic tail of growth factor receptors is the best example for this regulatory process, which serves as a docking platform for multiple effector molecules such as Grb2-Sos, phospholipase Cγ, p85–p110 subunits of phosphatidylinositol 3-kinase, GAP, and phosphotyrasine phosphatase (PTP).
KeywordsChloramphenicol Acetyl Transferase Restrictive Temperature Bacto Agar Library Plasmid Prey Protein