Abstract
Phosphorylation of two tyrosines in the death domain of CD95 is a critical mechanism in determining the receptor’s choices between cell death and survival signals. Recently, site-specific monoclonal antibodies against phosphorylated tyrosines of CD95 have been generated and used to successfully detect each phosphorylated death domain tyrosine of CD95 directly and separately by immunoblotting. Here we provide detailed protocols and useful tips for a successful site-specific detection of phosphorylated death domain tyrosine of CD95 following a protein separation by sizes (conventional SDS-PAGE) and by degrees of phosphorylation (phospho-protein affinity, mobility shift SDS-PAGE).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Chakrabandhu K et al (2007) Palmitoylation is required for efficient Fas cell death signaling. EMBO J 26(1):209–220
Chakrabandhu K et al (2016) An evolution-guided analysis reveals a multi-signaling regulation of Fas by tyrosine phosphorylation and its implication in human cancers. PLoS Biol 14(3):e1002401
Gradl G et al (1996) The CD95 (Fas/APO-1) receptor is phosphorylated in vitro and in vivo and constitutively associates with several cellular proteins. Apoptosis 1:131–140
Kinoshita E, Kinoshita-Kikuta E, Koike T (2009) Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE. Nat Protoc 4(10):1513–1521
Gordon JA (1991) Use of vanadate as protein-phosphotyrosine phosphatase inhibitor. Methods Enzymol 201:477–482
Mann M et al (2002) Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome. Trends Biotechnol 20(6):261–268
Strand S et al (2004) Cleavage of CD95 by matrix metalloproteinase-7 induces apoptosis resistance in tumour cells. Oncogene 23(20):3732–3736
Lajmanovich A et al (2009) Identification, characterisation and regulation by CD40 activation of novel CD95 splice variants in CD95-apoptosis-resistant, human, B-cell non-Hodgkin’s lymphoma. Exp Cell Res 315(19):3281–3293
Owen-Schaub L (2001) Soluble Fas and cancer. Clin Cancer Res 7(5):1108–1109
Leon-Bollotte L et al (2011) S-nitrosylation of the death receptor fas promotes fas ligand-mediated apoptosis in cancer cells. Gastroenterology 140(7):2009–2018, 2018.e2001–2004
Anathy V et al (2009) Redox amplification of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and S-glutathionylation of Fas. J Cell Biol 184(2):241–252
Feig C, Tchikov V, Schütze S, Peter ME (2007) Palmitoylation of CD95 facilitates formation of SDS-stable receptor aggregates that initiate apoptosis signaling. EMBO J 26(1):221–231
Kamitani T, Nguyen HP, Yeh ET (1997) Activation-induced aggregation and processing of the human Fas antigen. Detection with cytoplasmic domain-specific antibodies. J Biol Chem 272(35):22307–22314
Watanabe-Fukunaga R et al (1992) The cDNA structure, expression, and chromosomal assignment of the mouse Fas antigen. J Immunol 148(4):1274–1279
Itoh N et al (1991) The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66(2):233–243
Oehm A et al (1992) Purification and molecular cloning of the APO-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor receptor superfamily. Sequence identity with the Fas antigen. J Biol Chem 267(15):10709–10715
Shatnyeva OM et al (2011) Modulation of the CD95-induced apoptosis: the role of CD95 N-glycosylation. PLoS One 6(5):e19927
Keppler OT et al (1999) Differential sialylation of cell surface glycoconjugates in a human B lymphoma cell line regulates susceptibility for CD95 (APO-1/Fas)-mediated apoptosis and for infection by a lymphotropic virus. Glycobiology 9(6):557–569
García-Fuster MJ et al (2007) Effects of constitutive deletion of opioid receptors on the basal densities of Fas and Fas-associated protein with death domain (FADD) in the mouse brain: a delta-opioid tone inhibits FADD. Eur Neuropsychopharmacol 17(5):366–374
Chen L et al (2010) CD95 promotes tumour growth. Nature 465(7297):492–496
Peter ME (2014) DICE: a novel tumor surveillance mechanism-a new therapy for cancer? Cell Cycle 13(9):1373–1378
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Chakrabandhu, K., Huault, S., Hueber, AO. (2017). Site-Specific Detection of Tyrosine Phosphorylated CD95 Following Protein Separation by Conventional and Phospho-Protein Affinity SDS-PAGE. In: Legembre, P. (eds) CD95. Methods in Molecular Biology, vol 1557. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6780-3_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6780-3_16
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6778-0
Online ISBN: 978-1-4939-6780-3
eBook Packages: Springer Protocols