Functional Proteomics Mapping of a Human Signaling Pathway

Abstract

Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor β (TGFβ) superfamily. We used two-hybrid screening to map Smad signaling protein–protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.

Ubiquitination of Tumor Necrosis Factor Receptor-associated Factor 4 (TRAF4) by Smad Ubiquitination Regulatory Factor 1 (Smurf1) Regulates Motility of Breast Epithelial and Cancer Cells

“…Induces TRAF4 Ubiquitination-TRAF4 was identified previously by several groups as an interacting partner for Smurf1 and Smurf2 through yeast two-hybrid interaction screens (37,38,39). Subsequently, the in vivo interaction between TRAF4 and Smurf1 was confirmed through co-immunoprecipitation assays (38,39), with one group showing that Smurf1 was capable of inducing polyubiquitination of all six TRAF proteins (39).…”

PI3K, Erk Signaling in BMP7-Induced Epithelial-Mesenchymal Transition (EMT) of PC-3 Prostate Cancer Cells in 2- and 3-Dimensional Cultures

“…Survivin up-regulation by BMP-Smad signaling may be a common moderator of anti-apoptotic protection in prostate cancer cells. In this regard, Smad signaling pathway offers an important potential target for cancer [34][35][36].…”

TheShigella flexnerieffector OspG interferes with innate immune responses by targeting ubiquitin-conjugating enzymes

“…The ospG coding sequence was amplified by PCR and cloned into plasmid pB27 to screen the library constructed in plasmid pP6 by using random-primed cDNA made from human placenta poly(A) RNA, as described in ref. 18. The insert carried by prey plasmids in positive clones was amplified by PCR and sequenced to identify the corresponding gene in the GenBank database by using a fully automated procedure.…”