Mol Cell Proteomics
September 1, 2013;
The human PDZome: a gateway to PSD95-Disc large-zonula occludens (PDZ)-mediated functions.
Protein-protein interactions organize the localization, clustering, signal transduction, and degradation of cellular proteins and are therefore implicated in numerous biological functions. These interactions are mediated by specialized domains able to bind to modified or unmodified peptides present in binding partners. Among the most broadly distributed protein interaction domains, PSD95
-disc large-zonula occludens (PDZ) domains are usually able to bind carboxy-terminal sequences of their partners. In an effort to accelerate the discovery of PDZ domain interactions, we have constructed an array displaying 96% of the human PDZ domains that is amenable to rapid two-hybrid screens in yeast. We have demonstrated that this array can efficiently identify interactions using carboxy-terminal sequences of PDZ domain binders such as the E6 oncoviral protein and protein kinases (PDGFRβ, BRSK2
, and HER4); this has been validated via mass spectrometry analysis. Taking advantage of this array, we show that PDZ domains of Scrib
bind to the carboxy-terminal region of the planar cell polarity receptor Vangl2
. We also have demonstrated the requirement of Scrib
for the promigratory function of Vangl2
and described the morphogenetic function of SNX27
in the early Xenopus embryo
. The resource presented here is thus adapted for the screen of PDZ interactors and, furthermore, should facilitate the understanding of PDZ-mediated functions.
Mol Cell Proteomics
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Fig. 1. Human PDZome array to detect direct PDZ domain interactors.
A, schematic representation of the identification of interactions between the collection of PDZ domains and a protein of interest (POI) using a yeast two-hybrid assay. The PDZ domain collection cloned in the pAD vector is hosted by the yeast Y187 strain. This arrayed collection is probed with the desired POI cloned in the pDB vector and hosted by an AH109 strain. B, Coomassie-stained protein gel after pull-down assay on Caco2 and HEK 293T cell extracts using E6 or E6δTQL peptides as affinity reagents. C, graphic representation of Y2H (blue) and mass spectrometry (yellow) screens. Common identified E6 binders are in the green intersection of those two ensembles. In red, binders considered as indirect interactors as explained in the text.
Fig. 2. Interaction between E6 and selected PDZ domains measured by HTRF and ELISA.
A, shown in the table are the PDZ name, the molarity of the GST-PDZ fusion protein in purification eluates, Delta F (FRET intensity obtained with GST-PDZs, equal to 1E-8 M, determined as shown in B), IC50 (the apparent affinity determined via homologous competition assay as shown in C for representative PDZ domains (ND: not done)), and the binding intensity (A450) of the GST proteins (1E-7M) to solid phase E6 measured via ELISA, as shown in D (note the logarithmic scale of the y-axis). Green: high interaction; orange: low interaction; red: no interaction.
Fig. 4. Identification of PDZ partners of the Vangl2 carboxy-terminal region.
A, schematic representation of Vangl2 receptor. B, Coomassie-stained protein gel after pull-down assay on Caco-2 cell extracts using Vangl2 or Vangl2δTSV peptides as affinity reagents. Each star corresponds to PDZ interactors of the Vangl2 carboxy-terminal peptide found via mass spectrometry. In the right panel, an immunoblot from the same experiment using anti-Scrib antibody confirms the Vangl2–Scrib interaction. C, Scrib interacts with Vangl2 through its PDZ binding motif (PDZBM) sequence. HEK 293T cells were transfected with epitope-tagged Vangl2 or Vangl2 mutated at its PDZBM (last three amino acids mutated in alanine). Vangl2 proteins were purified using streptavidin beads, and the presence of Scrib was assessed by means of co-affinity purification and Western blotting. 15 μg of lysate were loaded in total lysate, and 400 μg were used for affinity purification. D, graphic representation of Y2H (blue) and mass spectrometry (yellow) screens. Common identified Vangl2 binders are in the green intersection of those two ensembles. In red are the binders considered as indirect interactors (see text).
Fig. 3. Screening of the PDZome with the PDZ binding site of protein kinases. A, carboxy-terminal sequences of selected protein kinases. B, silver-stained protein gel after pull-down assay on HEK 293T cell extracts using peptides from A as affinity reagents.
Fig. 5. Involvement of Scrib in Vangl2 promigratory function.
A, Western blot analysis of protein extracts of T47D cells stably transfected with GFP, GFP-Vangl2, or GFP-Vangl2Lp and GFP-Vangl2δTSV mutants. Degradation products of GFP-Vangl2Lp are marked by black points. B, each T47D cell population was submitted to transmigration assays in Boyden chambers without stimulation (blue bars) or with 1 nm HRG (red bars). Data are represented as fold changes and are the sum of three independent experiments. C, GFP-Vangl2 T47D cells either unstimulated or stimulated for 5 min with HRG treatment were fixed and labeled with anti-Scrib and Actin staining by TRITC-label and phalloidin, respectively. Confocal acquisition shows the localization of GFP-Vangl2 and Scrib at the leading edge under HRG stimulation. D, wild-type (Wt) MEF cells or circletail homozygous cells (Crc/Crc) were transiently transfected with GFP or GFP-Vangl2. The expression of proteins was evaluated with the mentioned antibodies. E, Wt and Crc/Crc MEF cells transiently transfected with GFP or GFP-Vangl2 were submitted to Boyden chamber assays using serum (FCS) as a chemoattractant. Filters were coated with fibronectin. Asterisks represent significant differences relative to the control (* p < 0.05 by Mann–Whitney U test). Data are represented as fold changes.
Fig. 6. SNX27 interacts with Vangl2 through its PDZ domain.
A, Western blot analysis of protein extracts of HEK 293T cells expressing STREP-HA-Vangl2 and either Myc-SNX27 or Myc-SNX27 deleted of its PDZ domain. Vangl2 proteins were purified using streptavidin beads, and the presence of SNX27 was assessed by means of co-affinity purification and Western blotting using anti-Myc antibody. 15 μg of lysate were loaded in total lysate, and 400 μg were used for affinity purification in A and B. B, SNX27 interacts with Vangl2 through its PDZBM sequence. HEK 293T cells were transfected with Vangl2 or Vangl2 mutated at its PDZBM (last three amino acids mutated in alanine). Vangl2 proteins were purified using streptavidin beads, and the presence of SNX27 (as indicated by an asterisk on the blot) was assessed by means of co-affinity purification and Western blotting. C, co-localization of Vangl2 with Myc-SNX27 but not Myc-SNX27δPDZ in transfected HEK 293T cells.
Fig. 7. SNX27 knockdown impairs morphogenetic movements in Xenopus embryos.
A, in situ hybridization with SNX27 antisense digoxygenin-labeled riboprobe revealed expression in early gastrula mesoderm (st. 10.5) and neural plate (st. 18). The st. 10.5 embryo on the right was bisected following staining to reveal expression in the internal mesodermal mantle (arrow). The st. 18 embryo is shown in front view (left) and dorsal view (right). Sense probe did not produce any signal. B, embryos were injected in dorsal blastomeres at 4- to 8-cell stage with SNX27 or Vangl2 morpholino-oligonucleotides (MOs) and stained for the neural marker Sox2 at the tailbud stage. Embryos representative of the three classes observed are shown. Class 1 embryos are wild-type, class 2 embryos display anterior neural tube opening (arrow), and class 3 embryos exhibit severe neural tube opening and a kinked body typical of deficient convergent extension. C, graph showing the phenotypic distribution of embryos injected with increasing amounts of SNX27 MOs. D, graph showing the synergistic effect of Vangl2 and SNX27 MOs.
snx27 (sorting nexin family member 27) gene expression in X. laevis embryo, NF stage 10.5, lateral view (image on left) and in bisected embryo (midsagittal section).
snx27 (sorting nexin family member 27) gene expression in X. laevis embryo, NF stage 18, anterior view, dorsal up ( image on left), and dorsal view, anterior left (image on right).
Anastas, A protein complex of SCRIB, NOS1AP and VANGL1 regulates cell polarity and migration, and is associated with breast cancer progression. 2012, Pubmed