Analysis of the PKC-δ Signaling Pathway by Proteomics with Embryonic and Genetic Engeneering

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The accumulation of genome sequence data has facilitated the establishment of new approaches to systematic characterization of gene expression profiles at the mRNA level (transcriptome). Such strategies do not, however, necessarily provide direct information about the profile of protein expression (proteome), given that the abundance of a given mRNA does not necessarily correlate with that of the encoded protein. Furthermore, numerous characteristics of proteins, including their subcellular localization, interactions with other molecules, stability, and posttranslational modification, are amenable to study only at the protein level. Recent advances in methods for protein identification based on MS and searches of protein or DNA sequence databases have allowed high-throughput analysis of the proteome. Posttranslational modification, including phosphorylation, regulates the functions of proteins by affecting their interactions with other molecules, their enzymatic activity, or their subc … More ellular localization and is pivotal to the control of many cellular processes. Such modification is difficult to identify by standard proteomics approaches, however, because the modified proteins usually constitute a small proportion of all protein molecules. It is therefore necessary first to concentrate such modified proteins and to prevent contamination by highly abundant proteins. Highly sensitive MS analysis is then able to detect various types of posttranslational modification. Protein kinase C (PKC), which comprises 11 closely related isoforms, has been implicated in a wide variety of signaling mechanisms. Among PKC isotypes, PKC-δ is unique in that its overexpression results in inhibition of cell growth. We showed that mice that lack PKC-δ exhibit enlargement of peripheral lymphoid organs, expansion of the B lymphocyte population, as well as the presence of numerous germinal centers in lymphoid tissues in the absence of stimulation. We tried to develop a new approach designated "Proteomics with Embryonic and Genetic Engineering (PGEM)" to uncover the changes in PKC-δ-null mice. In this study, we established efficient and large-scale methods for phosphorylation and ubiquitylation of proteins. Furthermore, we also appled the SILAC method to quantitative analysis of the phosphoproteome. Less