Most of the members of the superfamily of mammalian small heat shock or stress proteins are abundant in muscles where they play a role in muscle function and maintenance of muscle integrity. One member of this protein superfamily, human HSP27, is rapidly phosphorylated on three serine residues (Ser 15 , Ser 78 , and Ser 82 ) during cellular response to a number of extracellular factors. To understand better the role of HSP27, we performed a yeast two-hybrid screen of a human heart cDNA library for HSP27-interacting proteins. By using the triple aspartate mutant, a mimic of phosphorylated HSP27, as "bait" construct, a protein with a molecular mass of 21.6 kDa was identified as an HSP27-binding protein. Sequence analysis revealed that this new protein shares an overall sequence identity of 33% with human HSP27. This protein also contains the ␣-crystallin domain in its C-terminal half, a hallmark of the superfamily of small stress proteins. Thus, the new protein itself is a member of this protein superfamily, and consequently we designated it HSP22. According to the two-hybrid data, HSP22 interacts preferentially with the triple aspartate form of HSP27 as compared with wild-type HSP27. HSP22 is expressed predominantly in muscles. In vitro, HSP22 is phosphorylated by protein kinase C (at residues Ser 14 and Thr 63 ) and by p44 mitogen-activated protein kinase (at residues Ser 27 and Thr 87 ) but not by MAPKAPK-2.

The serine/threonine kinase protein kinase C ⑀ (PKC⑀) has been shown to be a critical component in the heart's resistance to cell death following ischemic insult. Recent studies have indicated that PKC⑀ forms multi-protein signaling complexes to accomplish signal transduction in cardiac protection. Using two-dimensional electrophoresis (2DE), combined with matrix-assisted laser desorption ionization mass spectrometry (MS), the initial analysis of these complexes identified signaling molecules, structural proteins, and stress-activated proteins. The initial analysis, although fruitful, was limited by the number of proteins revealed on the 2D gels. It was also apparent that many known cardiac protective functions of PKC⑀ could not be fully accounted for by the proteins identified in the initial analysis. Here we report the identification of an additional 57 proteins in PKC⑀ complexes using complimentary separation techniques, combined with high sensitivity MS. These techniques include 2DE or large format 1D SDS-PAGE followed by LC/MS/MS and solution trypsin digestion followed by LC/MS/MS, all of which yielded novel data regarding PKC⑀ protein complexes. Nanoscale LC/ MS/MS for the analysis of gel-isolated proteins was performed with sub-femtomole sensitivity. In contrast to 2DE analyses, the identification of proteins from 1D gels was independent of their visualization via staining and allowed for the identification of proteins with high isoelectric points. We found that PKC⑀ complexes contain numerous structural and signaling molecules that had escaped detection by our previous analyses. Most importantly, we identified two new groups of proteins that were previously unrecognized as components of the PKC⑀ complex: metabolism-related proteins and transcription/translation-related proteins. Molecular & Cellular Proteomics 1:421-433, 2002.