Rac1 activation controls nuclear localization of beta-catenin during canonical Wnt signaling

Abstract

Canonical Wnt signaling critically regulates cell fate and proliferation in development and disease. Nuclear localization of beta-catenin is indispensable for canonical Wnt signaling; however, the mechanisms governing beta-catenin nuclear localization are not well understood. Here we demonstrate that nuclear accumulation of beta-catenin in response to Wnt requires Rac1 activation. The role of Rac1 depends on phosphorylation of beta-catenin at Ser191 and Ser605, which is mediated by JNK2 kinase. Mutations of these residues significantly affect Wnt-induced beta-catenin nuclear accumulation. Genetic ablation of Rac1 in the mouse embryonic limb bud ectoderm disrupts canonical Wnt signaling and phenocopies deletion of beta-catenin in causing severe truncations of the limb. Finally, Rac1 interacts genetically with beta-catenin and Dkk1 in controlling limb outgrowth. Together these results uncover Rac1 activation and subsequent beta-catenin phosphorylation as a hitherto uncharacterized mechanism controlling canonical Wnt signaling and may provide additional targets for therapeutic intervention of this important pathway.

Figure 1

Rac1 activation is required for canonical Wnt signaling. (A-C) Western analyses to detect GTP-bound forms and total amounts of Rac1, Cdc42 and RhoA, in ST2 cells cultured in Wnt3a versus L conditioned medium (C. M.) for 0.5 or 1 hr. The relative amount of the GTP-bound form normalized to the total amount, in L medium, is designated 1.0. (D) Rac1 activation by purified recombinant Wnt3a protein (rWnt3a) at 50 ng/ml. (E) Expression of Lef1-Luciferase in cells infected with a control virus (IE, expressing GFP) or viruses expressing dnRac1 or dnCdc42. The cells were first infected with the viruses, then transiently transfected and cultured in regular growth medium for 47 hrs, and finally incubated in Wnt3a or L conditioned medium for 1 hr before being harvested. (F) Western analyses of Rac1 in cells at ∼96 hrs after transfection with control (Ctrl) or Rac1 siRNA. (G) Expression of Lef1-Luciferase following siRNA transfections. (H-I) Expression of AP following viral infection (H) or siRNA transfection (I). (J-K) Western analyses of β-catenin in cytosolic (J) or nuclear (K) fractions of cells cultured in L or Wnt3a medium for 1 hr following viral infections. Cytosolic and nuclear signals were normalized to GAPDH and CREB-1, respectively. *: p<0.05, n=3.

Figure 2

Mechanisms controlling Rac1 activation and canonical Wnt signaling. (A-B) Rac1 activation assays in cells cultured in L or Wnt3a medium for 1 hr following infection of indicated viruses. (C-D) Western analyses of cytosolic (C) versus nuclear β-catenin (D) in cells cultured in L or Wnt3a medium for 1 hr following infection of indicated viruses. Cytosolic and nuclear signals were normalized to GAPDH and CREB-1, respectively. (E) Lef1-luciferase expression by cells infected with indicated viruses and culture in L versus Wnt3a medium. (F) Western analyses of Dvl2 in ST2 cells transfected with Dvl2-specific or control siRNA. (G) Rac1 activation assays following Dvl2 knockdown. (H) Lef1-luciferase expression by cells transfected with gene-specific or control siRNA and culture in L versus Wnt3a medium. (I-J) Rac1 activation assays in cells infected with indicated viruses and cultured in L versus Wnt3a medium. (K) Effect of wortmannin on Rac1 activation at 1hr following Wnt3a stimulation in ST2 cells. (L) Effect of wortmannin on Lef1-Luciferase expression in ST2 cells. Transiently transfected cells were cultured in regular growth medium for 47 hrs, and then incubated in either L or Wnt3a conditioned medium (with or without 20 nM wortmannin) for 1 hr before being harvested. In all cases, wortmannin was added 3 hrs before and during the culture in conditioned media. (M) Western analyses of PIK3CA in cells transfected with gene-specific or control siRNA. *: p<0.05, n=3.

Figure 3

Rac1 activation enables Wnt7b to activate canonical Wnt signaling in ST2 cells. (A-B) Western analyses of β-catenin in cytosolic (A) or nuclear (B) fractions of ST2 cells following co-infection of viruses. Total viral titers were similar in each condition. (C) Rac1 activation assay in cells infected with control IE or Wnt7b virus. (D) Expression of Lef1-luciferase following co-infection. (E-G) Immunofluorescence confocal microscopy of cells following infection by the control IE virus alone (E), both control and Wnt7b viruses (F), or both caRac1 and Wnt7b viruses (G). The cells were infected at a similar titer. Nuclei were labeled green due to expression of nuclear GFP via IRES by all viruses used in the present study. β-catenin signal is in red. *: p<0.05, n=3.

Figure 4

JNK2 activation is required for canonical Wnt signaling. (A) Western analyses of phospho-JNK and total JNK in ST2 cells cultured in L versus Wnt3a medium for 0.5 or 1 hr. Phospho-JNK2 level was normalized to total JNK2. (B) Western analyses of phospho-JNK and total JNK in ST2 cells cultured in L versus Wnt3a medium for 1 hr following viral infections. (C) Effect of SP600125 on expression of Lef1-luciferase in ST2 cells. (D) Effect of SP600125 on expression of AP in ST2 cells. (E-F) Western analyses of β-catenin in cytolosic (E) or nuclear (F) fractions of ST2 cells cultured in L versus Wnt3a medium for 1 hr with varying concentrations of SP600125. SP600125 was added to the cells 3 hrs before and during the culture in conditioned media. (G) Effects of JNK1/2 knockdown on Lef1-Luciferase expression. Western analyses of JNK performed at ∼96 hrs after siRNA transfection. (H) Effects of JNK1/2 knockdown on nuclear localization of endogenous β-catenin as per immunofluorescence confocal microscopy. (I-K) Co-immunoprecipitation of endogenous β-catenin, JNK1/2 and Rac1 in cytosolic (I) versus nuclear (J) fractions of cells cultured in L versus Wnt3a medium for 1 hr (I-J) or infected with control (IE) or Wnt7b-expressing retrovirus. Purified IgG1 was used as control antibody for precipitation. *: p<0.05, n=3.

Figure 5

Rac1 and JNK activity are required for β-catenin nuclear localization in response to Wnt. ST2 cells were infected with indicated viruses and cultured in L or Wnt3a medium for 1 hr, with or without SP600125 (10μM), before being subjected to immunofluorescence confocal microscopy. (A-F) Nuclei as revealed by NLS-EGFP expressed by each virus via IRES. (A’-F’) Endogenous β-catenin detected by immunostaining. Yellow arrow in D’: enrichment of β-catenin at a cell-cell junction. White arrow in E’ and F’: enrichment of β-catenin in certain areas of the cell periphery. Note characteristic “fried-egg” cellular morphology in D’ and F’, caused by expression of caRac1. (A“-F”) Merged pictures of GFP and β-catenin signals.

Figure 6

Phosphorylation of Ser191 and Ser605 is critical for Wnt-induced nuclear localization of β-catenin. (A) Expression of Lef1-luciferase following transient transfection of β-catenin variants, and Western analyses of Myc-tagged β-catenin variants and endogenous β-catenin in the same lysates used for luciferase assays. (B) In vitro phosphorylation of Myc-tagged β-catenin variants by JNK2α2. Autoradiography signals were normalized to levels of Myc-β-catenin variants as determined by Western analyses using a Myc antibody. (C) In vivo phosphorylation of Myc-β-catenin variants in intact cells. Autoradiography signals were normalized to levels of Myc-β-catenin variants. (D) In vivo phosphorylation of Myc-β-catenin (wild type construct) in intact cells transfected with siRNA. Autoradiography signals were normalized to Myc-β-catenin levels. (E-L, E’-L’, E”-L”) Confocal images for nuclear GFP (E-L), exogenous myc-β-catenin variants (E’-L’) and the merge (E”-L”) in cells cultured with or without recombinant Wnt3a at 50 ng/ml for 1 hr, following infection with IE virus (expressing nuclear GFP) and transient transfection with plasmids expressing Myc-β-catenin variants.*: p<0.05, n=3.

Figure 7

Rac1 interacts genetically with β-catenin and Dkk1 in the limb bud ectoderm of mouse embryos. (A-D) Whole-mount skeletal preparations of E16.5 embryos. Black arrows denote complete absence of hindlimb structures in all Rac1-CKO embryos. The genotype for the control embryo is Msx2-Cre; Rac1^c/+. (A’-D’) Forelimb skeletons dissected from embryos above shown at a higher magnification. S: scapula; H: humerus; R: radius; U: ulna; P: phalanges. (E-F) Whole-mount Lac Z staining of E10 littermate embryos. Note that the control embryo (E) was partially squashed during staining so that the dorsal neural tube (red arrow) is now facing the reader instead of to the right. BA: 1^st branchial arch; FL: forelimb; HL: hindlimb. (E’, E”, F’,F”) Forelimb buds dissected from the embryos above shown at a higher magnification with either the dorsal (E’, F’) or the distal view (E”, F”). A: anterior; P: posterior; DE: distal ectoderm; DM: dorsal mesenchyme; VM: ventral mesenchyme. (G1-G2) H&E staining of forelimb sections through the AER region at E10.5. Boxed areas shown at a higher magnification. Red dotted line separates mesenchyme from ectoderm. Red arrow denotes AER. (H1-H2) Immunostaining of E-cadherin in the AER region of the forelimb at E10.5. Note thinner AER (red arrow) in the mutant. (I1-I2) TUNEL staining of forelimb sections at E10.5. Dotted white line demarcates ectoderm and AER. White arrow denotes TUNEL signal in the mutant AER. (J1-J2, K1-K2, L1-L2) Whole mount in situ hybridization of the forelimb at E10.5. Ventral view is shown for all limb buds, anterior to the left and posterior to the right. (M1-M3) Representative forelimbs (M1-M2) and hindlimb (M3, black arrow) of Msx1-Cre;Rac1^c/+;β-catenin^c/+ embryos at E16. (N1-N3) Representative forelimbs (N1-N2) and hindlimb (N3, black arrow) of Msx1-Cre;R26-Dkk1/R26-Dkk1 mice at birth. (O1-O3) Representative forelimbs (O1-O2) and hindlimb (O3, black arrow) of Msx1-Cre;Rac1^c/+;R26-Dkk1 embryos at E16.5. (P) A model for canonical Wnt signaling integrating β-catenin stabilization and Rac1-JNK2-mediated phosphorylation at Ser191 and Ser605 (small purple circles) necessary for β-catenin nuclear localization.