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. Tissue lysates were fractionated by ion exchange chromatography and used as substrates in in vitro kinase reactions. The phosphorylated proteins were separated using two-dimensional gel electrophoresis. Spots that contained isolated phosphoproteins were excised and digested with trypsin. The tryptic peptides were analyzed using mass spectrometry. While several of the proteins identified using this technique represent known PKC sub- strates, we identified a new PKC substrate in the initial screen. This protein, sm22, is expressed in smooth muscle cells and served well as a substrate for PKC in vitro. Sm22 is predominantly associated with the actin cytoskeleton. Upon activation of PKC in vivo, sm22 dissociates from the actin cytoskeleton and is distributed diffusely in the cytoplasm. Our data strongly suggest that phosphorylation by PKC controls the intra- cellular localization of sm22. This demonstrates that our approach, using a complex mixture of proteins as in vitro kinase substrates and subsequently identifying the newly phosphorylated proteins by mass spectrometry, is a powerful method to identify new ki- nase substrates. Keywords: Protein kinase C / Substrate / Two-dimensional gel electrophoresis / Mass spectro- metry EL 3926 Sascha Dammeier 1 Josip Lovric 1 Manfred Eulitz 1 Walter Kolch 2 J. Frederic Mushinski 3 Harald Mischak 4 1 Institut für Klinische Molekularbiologie, GSF München, Germany 2 CRC Beatson Laboratories, Glasgow, UK 3 Laboratory of Genetics, National Cancer Institute, National Institute of Health, Bethesda, MD, USA 4 Medizinische Hochschule Hannover, Germany 1 Introduction Protein kinase C (PKC) represents a family of ubiqui- tously expressed serine/threonine kinases. The family consists of ten known members in mammalian cells, which can be subdivided into three groups. The conven- tional cPKCs, a, bI, bII and g, fulfill all the criteria of a clas- sical PKC: their full activity is dependent on the presence of Ca 2+ as well as phospholipids and diacylglycerol. The novel PKCs, d, e, h, and q, do not require Ca 2+ and are activated by phospholipids and diacylglycerol alone. The atypical PKCs z and l(i) lack the diacylglycerol binding site, and their activation seems to depend on phospholi- pids only (reviewed in [1±3]). The ubiquitous expression of PKC isoforms and the high degree of structural conservation throughout many spe- cies indicate that PKCs play central roles in signal trans- duction processes. PKCs are known to be involved in processes such as differentiation, transformation, growth stimulation as well as growth inhibition, apoptosis as well as inhibition of apoptosis, migration, and cell motility (for reviews, see [1±3]). While some of these implications at first seem to be quite contradictory (transformation vs. growth inhibition, apoptosis vs. antiapoptotic signals), a common pattern emerges when the role of individual PKC isozymes is studied. For example, PKC-e induces trans- formation and protects from apoptotic stimuli in a variety of different cellular systems [4±6], while PKC-d inhibits cell growth and cell cycle progression; furthermore, acti- vation of this isozyme appears to represent a proapoptotic stimulus [4, 7, 8]. It is clear that the response of any cell to PKC activation depends on the precise combination of PKC isozymes expressed in that cell. Much insight has been gained by overexpression of active enzymes as well as by attempts to inhibit individual PKC isozymes by over- expression of inactive, dominant negative PKCs [9±13]. For a complete understanding of the molecular processes activated downstream of PKC activation in a given cell, detailed knowledge of the PKC substrates in this cell is essential. As we and others have shown, the PKC iso- zymes do not display a high degree of substrate specific- ity in vitro [14]. With few exceptions, the enzymes phos- phorylate identical proteins at the same sites and with Correspondence: Dr. Sascha Dammeier, Department of Molecular Biology, Byk Gulden Pharmaceuticals, Byk-Gulden- Straûe 2, D-78467 Konstanz, Germany E-mail: sascha.dammeier@byk.de Fax: +49-7531-843360 Abbreviations: GST, glutathione-S-transferase; PKC, protein kinase C; TPA, tetradecanoylphorbol-13-acetate; VSMC, vascu- lar smooth muscle cells Electrophoresis 2000, 21, 2443±2453 2443  WILEY-VCH Verlag GmbH, 69451 Weinheim, 2000 0173-0835/00/1212-2443 $17.50+.50/0 Proteomics and 2-DE