Introduction of Dominant Inhibitory Proteins Directed Against ROK and MRCK Kinases

Abstract

Gene-specific disruption and protein-specific inhibition techniques are powerful tools for the study of protein functions in vivo. A number of methods have been applied to investigate the loss of protein function. These include generation of knockout animals or cell lines, application of mRNA or protein ablation techniques, use of neutralizing antibodies or chemically synthesized inhibitory compounds (such as anti-sense oligonucleotides and enzyme inhibitors) and the important application of dominant-inhibitory mutants. The aim is to inhibit either the expression of genes of interest or the normal function of gene products. Here, we describe methods by which dominant interfering proteins for two related kinases can be expressed in mammalian cells, and show that the biological effects of inhibiting their respective signaling pathways are markedly different. Although dominant inhibitory GTPases mutants have provided a great deal of information about the specific pathways regulated by such GTPases, because of the plethora of effector proteins (in many cases), new reagents are required that can take into account each of these effectors in turn.