Valls G , Codina M , Miller RK , Del Valle-Pérez B , Vinyoles M , Caelles C , McCrea PD , García de Herreros A , Duñach M .

DK082145 NIDDK NIH HHS , HD07325 NICHD NIH HHS , R01 M52112 PHS HHS , T32 HD007325 NICHD NIH HHS , F32 DK082145 NIDDK NIH HHS , R01 GM052112 NIGMS NIH HHS

	Fig.1. Wnt-induced Rac1 activation and nuclear β-catenin translocation are dependent on p120-catenin. (A) SW-480 cells treated with control or Wnt3a-conditioned medium for the indicated time were lysed and active Rac1 was precipitated using a GST–PAK pull-down assay. Active Rac1 was detected by western blot (WB). All data are representative of at least five independent experiments. (B) The autoradiograms from experiments performed in triplicate as in A in SW-480 and HEK-293 cells were quantified and the mean ± s.d. was obtained for each condition. Each value is presented relative to that obtained in nonstimulated cells. (C) SW-480 cells stably expressing scrambled or shRNA specific for p120-catenin (p120-cat) were treated with control or Wnt3a-conditioned medium for 2 hours. GST–PAK pull-down assays were performed with control and depleted cell lysates and active Rac1 was determined by WB. (D) Autoradiograms from five different experiments performed in triplicate as in C were quantified and the mean ± s.d. was obtained for each condition. Each value is presented relative to that obtained in nondepleted cells treated with control medium. (E,F) Cytosolic and nuclear lysates were obtained from control and p120-catenin-depleted SW-480 (E) or HEK-293 (F) cells treated with control or Wnt3a-conditioned medium for 15 hours. The nuclear fraction was separated from the cytosolic and membrane-associated fraction as detailed in Materials and Methods. β-catenin levels in each cellular fraction were analyzed by WB. Lamin-β1 was used as a nuclear marker and pyruvate kinase as a marker for the cytosolic-plus-membrane fraction.
	Fig.2. Wnt-induced Rac1 activation is dependent on Vav2 interaction with p120-catenin. Vav2 (A) and p120-catenin (B) were immunoprecipitated from 500 µg SW-480 whole-cell extracts treated with control or Wnt3a-conditioned medium for the times indicated. Protein complexes were analyzed by western blot (WB) with anti-p120-catenin, anti-Vav2, anti-E-cadherin and anti-Rac1. 10 µg of SW-480 whole-cell extracts were included as internal reference (input). The graphs on the right are autoradiograms from four different experiments that were quantified and the mean ± s.d. obtained after 2 hours of incubation with Wnt3a medium. Each value is presented relative to that obtained in cells treated with control medium and normalized with respect to the amount of immunoprecipitated Vav2 or p120-catenin. (C) SW-480 cells stably expressing scrambled or shRNA specific for Vav2 were treated with control or Wnt3a-conditioned medium for 2 hours. GST–PAK pull-down assays were performed and active Rac1 was determined by WB. Autoradiograms from five different experiments performed in triplicate were quantified and the mean ± s.d. was obtained for each condition. Each value is presented relative to that obtained in nondepleted cells treated with control medium. (D) Cytosolic and nuclear lysates were obtained from control and Vav2-depleted SW-480 cells treated with control or Wnt3a-conditioned medium for 15 hours. β-catenin distribution between the two cell compartments was analyzed by WB.
	Fig.3. The direct interaction of p120-catenin with Rac1 and Vav2 is regulated by p120-catenin phosphorylation. (A) Vav2 was immunoprecipitated from 500 µg of control and p120-catenin (p120-cat)-depleted SW-480 cell extracts. Protein complexes were analyzed by western blot (WB) with anti-p120-catenin, anti-Rac1 and anti-Vav2. 5 µg of SW-480 whole-cell extracts were included as input. (B) GST–p120-catenin was phosphorylated by CK1, Src and Fyn at the times indicated and a pull-down assay was performed incubating 7 pmol of GST–p120-catenin or GST with cell extracts from SW-480 cells. Protein complexes were affinity purified and analyzed by WB. 4 µg of SW-480 lysate was included as input. (C) Full-length GST–p120-catenin wild type (wt), S 268,269 D (a p120-catenin mutant mimicking phosphorylated Ser268 and Ser269), or GST (2 pmol) were incubated with recombinant Rac1 (5 pmol). Protein complexes were affinity purified and analyzed by WB. (D) In vitro binding assays were performed by incubating GST–Rac1 or GST (5 pmol) with recombinant p120-catenin isoform 1 wt and p120-catenin mutants Y112E, Y217E (two Tyr→Glu mutants) or S268,269 D (Ser→Asp mutant) (1 pmol). Protein complexes were affinity purified and analyzed by WB with anti-p120-catenin and anti-GST mAbs. Internal reference standards (0.2 pmol) for p120-catenin wt and point mutants were included (St). (E) The isoform 1 of GST–p120-catenin wt and three specific point mutants (Y112E, Y217E and S268,S269D) (5 pmol) were incubated with HEK-293 cells lysates. Protein complexes were affinity purified and analyzed by WB with anti-Vav2 and anti-GST mAbs. 4 µg of HEK-293 lysate was included as input. (F) Autoradiograms from five different experiments performed in triplicate were quantified and the mean ± s.d. was obtained for each condition. The value of each mutant is presented relative to that obtained with p120-catenin WT construct. (G) p120-catenin was immunoprecipitated from 500 µg of SW-480 whole-cell extracts treated with control or Wnt3a-conditioned medium for 2 hours. 2 µg of SW-480 whole-cell extracts was included as input. Protein complexes were analyzed by WB. (H) Fyn was immunoprecipitated from 500 µg SW-480 whole-cell extracts treated with control or Wnt3a-conditioned medium for 2 hours. Protein complexes were analyzed by WB. 5 µg of SW480 lysate was included as input. (I) GST–p120-catenin (5 pmols) was phosphorylated by CK1 and incubated with SW-480 cell extracts. Protein complexes were affinity purified and analyzed by WB. 5 µg of SW-480 lysate was included as input.
	Fig.4. Only p120-catenin mutants able to interact with Rac1 and Vav2 induce Rac1 activation. (A) GST–PAK pull-down assays were performed in SW-480 cell extracts overexpressing either GFP–p120-catenin (p120-cat) wild-type (wt) isoforms 1 or 3, GFP–p120-catenin 1 harboring point mutants Y112E or Y217E, GFP–p120-catenin deletion mutants (350–911) or (1–234) or the empty vector phrGFP. Active Rac1 was detected by western blot (WB). (B) Autoradiograms from five different experiments performed in triplicate in SW-480 (left panel) and HEK-293 cells (right panel) were quantified and the mean ± s.d. was obtained for each condition. Each value is presented relative to that obtained in nonstimulated cells. (C–F) Cytosolic and nuclear lysates were obtained from HEK-293 (C,D) or SW-480 (E,F) cells overexpressing either GFP–p120-catenin wt isoform 1, GFP–p120-catenin point mutants Y112E or Y217E or the empty vector phrGFP, treated when indicated with Wnt3a-conditioned medium for 15 hours. The nuclear fraction was separated from the cytosolic and membrane-associated fraction as detailed in Materials and Methods. β-catenin levels in each cellular fraction were analyzed by WB. Lamin-β1 was used as a nuclear marker and pyruvate kinase as a marker for the cytosolic-plus-membrane fraction. In the right panel of C, p120-catenin wt, Y112E and Y217E expression levels were analyzed by WB. (D,F) Autoradiograms from four (F) or five (D) different experiments were quantified and the mean ± s.d. was obtained for each condition. Each value is presented relative to that obtained in nonstimulated and nontransfected cells.
	Fig.5. p120-catenin mutants unable to interact with Rac1 and Vav2 compromise Rac1 activation but not earlier steps of Wnt signaling. (A) E-cadherin was immunoprecipitated from control and p120-catenin (p120-cat)-depleted SW-480 cells overexpressing GFP–p120-catenin wild-type (wt) isoform 1, GFP–p120-catenin 1 point mutants Y112E, Y217E, S268,269A or the empty vector phrGFP and E-cadherin, and treated with control or Wnt3a-conditioned medium for 2 hours. Protein complexes were analyzed by western blot (WB) with the antibodies indicated. (B) Control and p120-catenin depleted HEK-293 cells overexpressing GFP–p120-catenin wt isoform 1, GFP–p120-catenin point mutants Y112E, Y217E and S268,269A or the empty vector phrGFP were treated with control or Wnt3a-conditioned medium for 9 hours and total β-catenin levels were analyzed by WB. (C) Autoradiograms from five different experiments performed as in B were quantified and the mean ± s.d. was obtained for each condition. Each value is presented relative to that obtained in nondepleted and nonstimulated cells. (D,E) p120-catenin-depleted SW-480 (D) or HEK-293 (E) cells overexpressing GFP–p120-catenin wt isoform 1, GFP–p120-catenin 1 harboring point mutants Y112E, Y217E, S268,269A and E-cadherin or the empty vector phrGFP were treated with control or Wnt3a-conditioned medium for 2 hours. GST–PAK pull-down assays were performed and active Rac1 was detected by WB. The graphs on the right show autoradiograms from five different experiments performed in triplicate) that were quantified and the mean ± s.d. obtained for each condition. Each value is presented relative to that obtained in nondepleted and nonstimulated cells.
	Fig.6. Only cytosolic p120-catenin interacts with Vav2 and activates Rac1. (A) HEK-293 cells were transfected with GFP–p120-catenin (p120-cat) isoform 1 point mutants S268,269A, S268,269D and E-cadherin or the empty vector phrGFP. GST–PAK pull-down assays were performed and active Rac1 was detected by western blot (WB). (B) Autoradiograms from five different experiments performed in triplicate as in A in SW-480 and HEK-293 cells were quantified and the mean ± s.d. was determined for each condition. Each value is presented relative to that obtained in nontransfected cells. (C) Cytosolic and membrane lysates were prepared from HEK-293 cells coexpressing GFP–p120-catenin 1 harboring point mutants S268,269A or S268,269D and E-cadherin or the empty vector. The subcellular distributions of p120-catenin mutants, Vav2, E-cadherin and Rac1 were analyzed by WB. Pyruvate kinase was used as a marker for the cytosolic fraction. (D) Vav2 was immunoprecipitated from 500 µg HEK-293 whole-cell extracts transfected with either E-cadherin or the empty vector. Protein complexes were analyzed by WB with anti-p120-catenin and anti-Vav2. 5 µg of HEK-293 whole-cell extracts was included as input. (E) GST–p120-catenin isoform 1 or GST (1.5 pmol) were incubated with recombinant Vav2 (3 pmol) in the presence of a tenfold molecular excess of cyto-E-cadherin (15 pmol) when indicated. Protein complexes were affinity purified and analyzed by WB with the indicated mAbs. Vav2 (0.3 pmol) was included as an internal reference standard (St). (F) p120-catenin was immunoprecipitated from 500 µg HEK-293 whole-cell extracts transfected with either E-cadherin or the empty vector. Protein complexes were analyzed by WB with anti-Vav2, anti-E-cadherin, anti-phospho-tyrosine and anti-p120-catenin. 5 µg of HEK-293 whole-cell extracts was included as input. The autoradiograms in D–F correspond to the four different experiments performed; the autoradiograms were quantified and the mean ± s.d. was obtained for each condition. The result obtained after E-cadherin expression was represented relative to the control.
	Fig.7. p120-catenin point mutants unable to bind Rac1 or Vav2 do not rescue gastrulation defects caused by p120-catenin depletion in Xenopus embryos. Embryos at the two-cell stage were injected with a control morpholino or a combination of xp120 morpholinos (20 ng each of xp120 MO I and MO II per blastomere). Rescues were performed by co-injection of xp120 morpholinos with in vitro transcribed mRNAs corresponding to mp120-catenin (mp120-cat) WT, mp120-catenin Y112E and mp120-catenin Y112F (A,B), or mp120-catenin Ser268,269A and mp120-catenin Ser268,269D (C,D). Embryos were scored at stage 12 (gastrula) for defects, including partial or improper closure of the blastopore. Representative resulting gastrulation phenotypes are shown in A and C, with percentages presented in B and D, including error bars. To avoid overexpression phenotypes, the injection doses of all rescuing constructs were carefully titrated to produce minimal effects when injected alone. (E) Embryos at the two-cell stage were injected with a control morpholino or a combination of xp120 morpholinos (20 ng each of xp120 MO I and MO II per blastomere). Rescues were performed by co-injection of xp120 morpholinos with mp120-catenin Y112F, CA-Rac, p120-catenin Y112E and mp120-catenin Y217E in vitro transcribed mRNAs. A small-rescue dose of CA-Rac1 was also co-injected with mp120 Y112E and mp120 Y217E constructs. Percentage of gastrulation delay from three different experiments is shown in (F). P values were determined using ANOVA.
	Fig.8. Model for Wnt-induced Rac1 activation through p120-catenin and Vav2. In nonstimulated cells (A), p120-catenin (p120-cat) is bound to E-cadherin (E-cadh), and through this protein to LRP5/6. Upon binding of the Wnt factor (B), a receptor complex is formed that includes p120-catenin, E-cadherin, LRP5/6, Axin, Dvl and Frizzled (Fz). (C) p120-catenin is released from E-cadherin upon its phosphorylation in Ser268 and Ser269 by CK1α. This phosphorylation also decreases Src/Fyn interaction with p120-catenin and consequently promotes dephosphorylation of tyrosine residues in the p120-catenin regulatory domain. Ser-phosphorylated, Tyr-dephosphorylated p120-catenin presents a higher affinity for Vav2 and Rac1 (D). Association with p120-catenin enhances Vav2 activity towards Rac1 and activates this GTPase. GTP-bound Rac1 stimulates PAK kinase that phosphorylates and activates JNK2 (E). Both JNK2 and PAK phosphorylate β-catenin previously released from the adherens junction, enabling the translocation of this protein to the nucleus (F). Another pool of p120-catenin also travels to the nucleus, where it relieves the inhibition caused by the Kaiso transcriptional repressor (Del Valle-Pérez et al., 2011b).

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