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.
Highlights
Multiprotein complexes play an essential role in the propagation and integration of cellular signals
The peptides were selected as a set of known binding motifs for interaction domains of proteins involved in T-cell signal transduction with a focus on interactions up- and downstream of the scaffolding protein LAT (Supplemental Table 1)
Phosphorylation-dependent Changes in Interaction Profiles—The formation of protein complexes upon tyrosine phosphorylation plays a major role in T-cell signaling
Summary
Multiprotein complexes play an essential role in the propagation and integration of cellular signals. For interactions of SH2 domain-containing proteins on Tyr(P) peptides representing known binding partners, pervanadate induced a moderate signal decrease as for example for GADS, GRB2, and PLC␥1 on the respective pepLATpY motifs. For proteins directly binding to cognate poly(Pro) motifs through SH3 domains, we observed only moderate signal changes, often minor increases, as for example for PLC␥1 binding to pepSLP179 or GRB2 binding to pepGAB2-509 [21] (for the nomenclature of peptides refer to Supplemental Table 1).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
