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Quantitative proteome analysis of detergent‐resistant membranes identifies the differential regulation of protein kinase C isoforms in apoptotic T cells
Author(s) -
Solstad Therese,
Bjørgo Elisa,
Koehler Christian J.,
Strozynski Margarita,
Torgersen Knut Martin,
Taskén Kjetil,
Thiede Bernd
Publication year - 2010
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201000164
Subject(s) - jurkat cells , apoptosis , protein kinase c , microbiology and biotechnology , flow cytometry , biology , proteome , lipid raft , signal transduction , pkc alpha , biochemistry , t cell , immunology , immune system
Abstract Several lines of evidence suggest that detergent‐resistant membranes (DRMs) (also known as lipid rafts and glycosphingolipid‐enriched microdomains) may have a role in signaling pathways of apoptosis. Here, we developed a method that combines DRMs isolation and methanol/chloroform extraction with stable isotope labeling with amino acids in cell culture‐based quantitative proteome analysis of DRMs from control and cisplatin‐induced apoptotic Jurkat T cells. This approach enabled us to enrich proteins with a pivotal role in cell signaling of which several were found with increased or decreased amounts in DRMs upon induction of apoptosis. Specifically, we show that three isoforms of protein kinase C (PKC) are regulated differently upon apoptosis. Although PKCα which belongs to the group of conventional PKCs is highly up‐regulated in DRMs, the levels of two novel PKCs, PKCη and PKCθ, are significantly reduced. These alterations/differences in PKC regulation are verified by immunoblotting and confocal microscopy. In addition, a specific enrichment of PKCα in apoptotic blebs and buds is shown. Furthermore, we observe an increased expression of ecto‐PKCα as a result of exposure to cisplatin using flow cytometry. Our results demonstrate that in‐depth proteomic analysis of DRMs provides a tool to study differential localization and regulation of signaling molecules important in health and disease.