Profiling substrate phosphorylation at the phosphopeptide level.

The identification of substrates is a key aspect in the study of the biological function of protein kinases. The procedure here described is aimed at profiling substrate phosphorylation at the phosphopeptide level by sequentially involving (i). the assessment of the in vitro activity of individual protein kinases on a complex mix of immobilized proteins, (ii). the fractionation of the phosphopeptides being released upon proteolysis of substrates, and (iii). the final identification of the targeted sequences. In particular, the protein sample is spotted onto nitrocellulose membrane and then subjected to a solid-phase kinase assay in the presence of [32P]ATP, prior to solid-phase proteolytic digestion and two-dimensional phosphopeptide mapping. Radiolabeled phosphopeptides are subsequently isolated and sequenced to identify the substrates being targeted by the examined protein kinase. Using the gamma-isotype of p21-activated protein kinase (gamma-PAK) and its known in vitro substrates, I verified that both the specificity of substrate phosphorylation and its efficiency are similar upon solid- and liquid-phase conditions. To demonstrate the feasibility of the overall experimental system, I then employed a fairly crude cell extract as a source of candidate substrates and successfully identified the sequence of a putative substrate of gamma-PAK.