A double-labeling method for measuring induction of protein phosphorylation.

Protein phosphorylation is widely believed to play a regulatory role in signal transduction, mitosis, cell proliferation, cell motility, cell shape, gene regulation, and many other cellular processes. Thus, the quantitation of phosphorylation of specific cellular proteins may provide insight into the mechanisms by which phosphorylation is employed in regulation. Moreover, identification of phosphorylation substrates of various cellular kinases provides an important first step in determining their role in cellular regulation. However, accurate measurement of the differential phosphorylation of cellular proteins under different physiological conditions is often difficult to achieve. To address this problem, we have developed an in vivo double-labeling protocol (utilizing [3H]-, [14C]-, or [35S]-radiolabeled amino acids and [32P]-orthophosphate) that allows the quantitation of the amount of specific phosphorylation of a given protein from densitometric analysis of autoradiograms of polyacrylamide gels. This double-labeling strategy provides a means of quantitating the phosphorylation of individual biosynthetically labeled proteins. This method can be used in the analysis of immunoprecipitated proteins, proteins from subcellular fractions, such as nuclei or selected membrane fractions, or even total cellular proteins displayed on two-dimensional gels.