Analysis of Protein Phosphorylation in Intact Cells and Extracts

The role of protein phosphorylation in regulating and integrating the activity of the nervous system has become widely appreciated during the last few decades. Both extracellular signals (e.g., neurotransmitters, hormones, growth factors, and electrical activity of the nerve cell itself) and intracellular signals and effecters (e.g., cyclic nucleotides, Ca²⁺ ions and phospholipid derivatives) mediate many of their effects by regulating the activities of protein phosphorylation systems (Walaas and Greengard, 1991). A variety of methods have been developed to investigate these systems (Rudolph and Krueger, 1979; Palfrey and Mobley, 1987), many of which have been recently described in detail (Hunter and Sefton, 1991a,b). Here we will describe a number of well-established methods that have been used to investigate protein phosphorylation systems in intact cells and extracts from neural tissue, with emphasis placed on methods suitable for studies on vertebrate preparations. Complete coverage of the literature is not intended, and only a limited number of methods that have proven useful in our laboratory will be presented.