Identification of the smooth muscle-specific protein, sm22 as a novel protein kinase C substrate using two-dimensional gel electrophoresis and mass spectrometry

We report a novel method to identify protein kinase C (PKC) substrates. Tis sue lysates were fractionated by ion exchange chromatography and used as su bstrates in in vitro kinase reactions. The phosphorylated proteins were sep arated using two-dimensional gel electrophoresis. Spots that contained isol ated phosphoproteins were excised and digested with trypsin. The tryptic pe ptides were analyzed using mass spectrometry. While several of the proteins identified using this technique represent known PKC substrates, we identif ied a new PKC substrate in the initial screen. This protein, sm22, is expre ssed in smooth muscle cells and served well as a substrate for PKC in vitro . Sm22 is predominantly associated with the actin cytoskeleton. Upon activa tion of PKC in vivo, sm22 dissociates from the actin cytoskeleton and is di stributed diffusely in the cytoplasm. Our data strongly suggest that phosph orylation by PKC controls the intracellular localization of sm22. This demo nstrates that our approach, using a complex mixture of proteins as in vitro kinase substrates and subsequently identifying the newly phosphorylated pr oteins by mass spectrometry, is a powerful method to identify new kinase su bstrates.