ISBN 9783843901307

978-3-8439-0130-7, Reihe Biochemie

Roland Lehmann
Development of a novel S3S-TAP-tag for a proteomic interactome analysis of the human T cell adaptor protein ADAP

171 Seiten, Dissertation Freie Universität Berlin (2011), Hardcover, A5

Zusammenfassung / Abstract

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.