Quantitative proteome analysis of detergent-resistant membranes identifies the differential regulation of protein kinase C isoforms in apoptotic T cells.
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 PKC alpha which belongs to the group of conventional PKCs is highly up-regulated in DRMs, the levels of two novel PKCs, PKC eta and PKC theta, are significantly reduced. These alterations/differences in PKC regulation are verified by immunoblotting and confocal microscopy. In addition, a specific enrichment of PKC alpha in apoptotic blebs and buds is shown. Furthermore, we observe an increased expression of ecto-PKC alpha 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.
Solstad
Therese
T
The Biotechnology Centre of Oslo University of Oslo, Oslo, Norway.
Bjørgo
Elisa
E
Koehler
Christian J
CJ
Strozynski
Margarita
M
Torgersen
Knut Martin
KM
Taskén
Kjetil
K
Thiede
Bernd
B
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002458:Cell Fractionation; D002945:Cisplatin / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D021962:Membrane Microdomains / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013601:T-Lymphocytes / Q000166:cytology* / Q000201:enzymology
D000970:Antineoplastic Agents; D020033:Protein Isoforms; D020543:Proteome; D011493:Protein Kinase C; D002945:Cisplatin