Analysis of cellular phosphoproteins by two-dimensional gel electrophoresis: applications for cell signaling in normal and cancer cells.

Two-dimensional (2-D) gel electrophoresis has been used to map proteins from various cell types in an effort to eventually link such maps to the sequencing of the entire human genome. While this analysis indicates the cellular disposition and expression of proteins, another application of 2-D gels, the analysis of phosphoproteins, can provide much information as to the assembly and "wiring" of the signal transduction circuits within cells which appear to be enervated by phosphate exchange. The preparation and separation of 32P-labeled proteins is described, as well as various analytical methods, including: the variety of gel systems available for specialist types of analyses, comparing 33P- and 32P-labeling of proteins, imaging techniques, phosphoamino analysis, phosphopeptide separation, identifying the amino acid groups that are phosphorylated, and the identification of phosphoproteins on 2-D gels by immunoprecipitation, corunning of purified proteins, comparative mapping and microsequencing, and by Western blotting. Examples (in brackets) are given of applications in which 2-D phosphogels can be applied, which offer advantages over other techniques. These include: (i) identifying in vivo substrates for kinases (protein kinase C activated by phorbol myristate acetate), (ii) investigating cytokine signaling pathways (tumor necrosis factor and interleukin-1), (iii) investigating the effects of drugs on signaling pathways (okadaic acid, menadione and cyclooxygenase inhibitors), (iv) characterization of specific phosphoproteins (heat-shock protein Hsp27 and stathmin), (v) comparing normal and transformed cells (MRC-5 human lung fibroblasts and their SV-40-transformed counterparts, MRC-5 SV1 cells), (vi) purifying phosphoproteins, (vii) investigating the relationship of protein phosphorylation to stages in the cell cycle (stathmin), (viii) investigating protein/protein interactions, (ix) mapping in vitro kinase substrates (protein kinase C, protein kinase A, and mitogen activated protein kinase activated protein kinase 2), and (x) locating and identifying cellular phosphatases (Hsp27 phosphatase). It is possible that the mapping of phosphoproteins can be linked to other 2-D gel databases and that information derived from these can be used in the future to better understand the signaling mechanisms of normal and cancerous cells.