Development of a novel S3S-TAP-tag for a proteomic interactome analysis of the human T cell adaptor protein ADAP
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Beschreibung
This work has made important strides to clarify the functions of the T cell scaffolding protein ADAP beyond its known role in TCR induced "inside-out" signaling. The novel TAP-tag technique enabled a full interactome analysis of ADAP which, together with results from other research groups, extend its role by uncovering links to the cytoskeleton, the nucleus and also to “outside-in” signaling.
The developed S3S-tag in combination with a stable genome incorporation of S3S-tagged ADAP to sort for a uniform protein expression proved to be successful for the isolation of mammalian protein complexes, as it enabled the complete analysis of protein interaction partners of ADAP in resting T cells. Furthermore, the application of SILAC-MS allowed for quantification of isolated proteins and to discriminate likely candidates of physiologically bound proteins.
Further evidence for a nuclear localization and function was provided by confocal microscopy and cellular fractionation analysis, which propose a nuclear pool of ADAP. A functional link is proposed by the identification of several ADAP associated proteins involved in transcriptional and translational control of IL-2 and NFkB.
Furthermore, two unexplored serine phosphorylation sites in ADAP were confirmed by mass spectrometry.
This work has made important strides to clarify the functions of the T cell scaffolding protein ADAP beyond its known role in TCR induced "inside-out" signaling. The novel TAP-tag technique enabled a full interactome analysis of ADAP which, together with results from other research groups, extend its role by uncovering links to the cytoskeleton, the nucleus and also to “outside-in” signaling.
The developed S3S-tag in combination with a stable genome incorporation of S3S-tagged ADAP to sort for a uniform protein expression proved to be successful for the isolation of mammalian protein complexes, as it enabled the complete analysis of protein interaction partners of ADAP in resting T cells. Furthermore, the application of SILAC-MS allowed for quantification of isolated proteins and to discriminate likely candidates of physiologically bound proteins.
Further evidence for a nuclear localization and function was provided by confocal microscopy and cellular fractionation analysis, which propose a nuclear pool of ADAP. A functional link is proposed by the identification of several ADAP associated proteins involved in transcriptional and translational control of IL-2 and NFkB.
Furthermore, two unexplored serine phosphorylation sites in ADAP were confirmed by mass spectrometry.
Autor*in
Roland Lehmann