A novel S3S-TAP-tag for the isolation of T-cell interaction partners of adhesion and degranulation promoting adaptor protein.
The identification of modular units of cellular function is a major goal for proteomic research. Protein complexes represent important building blocks defining functionality and deciphering their composition remains a major challenge. Here, we have designed a new tandem affinity purification (TAP) tag (termed S3S-tag) for the isolation of protein complexes. Specifically, the immune cell protein ADAP that regulates integrin adhesion was fused either C- or N-terminally to the S3S-tag. After retroviral transduction of a vector containing S3S-tagged ADAP and internal ribosomal entry site encoded enhanced green fluorescent protein (eGFP), Jurkat T cells were sorted according to eGFP expression and further selected for expression of TAP-tagged protein close to endogenous levels. The combination of a cleavable S-tag and a Strep-tag II allowed for the isolation of ADAP and associated proteins. Subsequently, stable isotope labeling with amino acids in cell culture-based mass spectrometric analysis was performed to identify potentially specific interaction partners. Co-purification of the known interaction partner Src kinase-associated phosphoprotein of 55 kDa indicates the validity of our approach, while the identification of the ENA/VASP family member EVL, the guanine nucleotide exchange factor GEF-H1 and the adaptor protein DOCK2 corroborates a link between ADAP-mediated integrin regulation and the cytoskeleton.
Lehmann
Roland
R
Leibniz-Institute for Molecular Pharmacology, Berlin, Germany.
Meyer
Johann
J
Schuemann
Michael
M
Krause
Eberhard
E
Freund
Christian
C
D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005434:Flow Cytometry; D020690:GTPase-Activating Proteins; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D020662:Guanine Nucleotide Exchange Factors / Q000032:analysis / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D064067:Rho Guanine Nucleotide Exchange Factors; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D014162:Transfection
C577356:ARHGEF2 protein, human; D048868:Adaptor Proteins, Signal Transducing; D015815:Cell Adhesion Molecules; C402110:DOCK2 protein, human; C484449:EVL protein, human; C106957:FYB1 protein, human; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D010750:Phosphoproteins; D064067:Rho Guanine Nucleotide Exchange Factors; C108336:SKAP1 protein, human