title: A network analysis of changes in molecular interactions in cellular signaling.
abstract: Multiprotein complexes play an essential role in the propagation and integration of cellular signals. However, systems level analyses of signaling-dependent changes in the pattern of molecular interactions are still missing. Signaling in T-lymphocytes is one prominent example in which multiprotein complexes orchestrate signal transduction. We implemented peptide microarrays comprising a set of interaction motifs of signaling proteins for network-based analyses of signaling-dependent changes in molecular interactions. Lysates of resting or stimulated cells were incubated on these arrays, and the binding of signaling proteins was detected by immunofluorescence. Signaling-dependent complex formation led to changes of signals on the microarrays in two ways. 1) Masking of a binding site of a signaling protein for a peptide on the array resulted in a signal decrease. 2) Interaction of a protein with a second protein, which in turn binds to a peptide on the array, resulted in a signal increase for the first protein. Dissipation of complexes led to the reverse changes. Competition with peptides corresponding to interaction motifs provided detailed information on the architecture of complexes; lack of individual signaling proteins revealed the functional interdependence of interactions in the network. We show that complex formation through phosphorylation of the scaffolding protein LAT (linker for activation of T-cells) acted as a signal amplifier. PLCgamma1 deficiency increased the resting state levels of LAT-dependent complexes and augmented the recruitment of the phosphatase SHPTP2 into complexes. For the analysis of signaling networks, the parallel detection of changes in interactions enabled the identification of functional interdependencies with minimum a priori knowledge.
authors.affiliation: Interfaculty Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany.
authors.forename: Oda
authors.initials: O
authors.lastname: Stoevesandt
authors.forename: Karsten
authors.initials: K
authors.lastname: Köhler
authors.forename: Susann
authors.initials: S
authors.lastname: Wolf
authors.forename: Thomas
authors.initials: T
authors.lastname: André
authors.forename: Wilfred
authors.initials: W
authors.lastname: Hummel
authors.forename: Roland
authors.initials: R
authors.lastname: Brock
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008565:Membrane Proteins / Q000378:metabolism; D010455:Peptides / Q000032:analysis*; D051966:Phospholipase C gamma / Q000378:metabolism; D010766:Phosphorylation; D040081:Protein Array Analysis; D025941:Protein Interaction Mapping; D015398:Signal Transduction
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C110810:LAT protein, human; D008565:Membrane Proteins; D010455:Peptides; D051966:Phospholipase C gamma