Methods for measuring RGS protein phosphorylation by G protein-regulated kinases

Little is known about cellular regulation of the regulators of G protein signaling (RGS) proteins, principal players in G protein signaling. These proteins are known for their capacity to negatively regulate G protein signals, however, their chief cellular functions may expand beyond this limited role. Comprehensive understanding of cellular roles of RGS proteins requires knowledge of their regulation by short latency and inducible signals, such as kinase activation by G proteins. A number of RGS proteins are phosphorylated in cells, with varied effects on their function and localization. These studies focus on RGS14, which contains recognition motifs for several G protein-regulated kinases. Procedures used in our laboratory to study the phosphorylation of RGS14 are outlined, and the method used to purify RGS14 is described with notes on complications that may be encountered. Standard protocols used to investigate the recognition of RGS proteins by 3-5-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), extracellular signal-regulated kinase (ERK), and protein kinase C (PKC) are described, followed by strategies used to establish the specific amino acids modified by these kinases. Although this chapter focuses on investigations into RGS14, the protocols described are readily modified for other RGS proteins.