Integrating patterning signals: Wnt/GSK3 regulates the duration of the BMP/Smad1 signal

Abstract

BMP receptors determine the intensity of BMP signals via Smad1 C-terminal phosphorylations. Here we show that a finely controlled cell biological pathway terminates this activity. The duration of the activated pSmad1(Cter) signal was regulated by sequential Smad1 linker region phosphorylations at conserved MAPK and GSK3 sites required for its polyubiquitinylation and transport to the centrosome. Proteasomal degradation of activated Smad1 and total polyubiquitinated proteins took place in the centrosome. Inhibitors of the Erk, p38, and JNK MAPKs, as well as GSK3 inhibitors, prolonged the duration of a pulse of BMP7. Wnt signaling decreased pSmad1(GSK3) antigen levels and redistributed it from the centrosome to cytoplasmic LRP6 signalosomes. In Xenopus embryos, it was found that Wnts induce epidermis and that this required an active BMP-Smad pathway. Epistatic experiments suggested that the dorsoventral (BMP) and anteroposterior (Wnt/GSK3) patterning gradients are integrated at the level of Smad1 phosphorylations during embryonic pattern formation.

Figure 1. Smad1 Is Phosphorylated and Inhibited by GSK3

(A) Smad1 contains MAPK (blue) and GSK3 (red) phosphorylation sites in its linker region. The PPAY binding site of Smurf1 is boxed and serines 210 and 214 used to raise antibodies indicated by asterisks. (B) Smad1 constructs encoding Smad1 wild-type (SWT) or phosphorylation-resistant mutants for GSK3 (SGM) and MAPK (SMM) sites. (C–F) Injection of SGM or SMM, but not of SWT mRNA, increased expression of the ventral marker sizzled in Xenopus embryos. (G) A BMP-independent phospho-mimetic activated Smad1 (SEVE) in which the SVS terminus was mutated into EVE. (H–J) Activity of SEVE is increased by phosphorylation-resistant linker mutations. (K) GSK3 radioactively phosphorylates Smad1 in vitro, but only when primed by MAPK. The recombinant Smad1 linker substrate was about 90% pure in polyacrylamide gels (data not shown). (L and M) Phospho-specific antibodies for hSmad1 Ser 214 (pSmad1^MAPK) and Ser 210 (pSmad1^GSK3 antibody B was used). (N) Phospho-specific antibodies (pSmad1^MAPK and pSmad1^GSK3-A) demonstrate that GSK3 phosphorylation of recombinant Smad1 requires MAPK priming, in non-radioactive western blots. Recombinant Smad1 substrate was used in the same amount as in panel K.

Figure 2. GSK3 and MAPK Phosphorylation Sites Regulate Polyubiquitinylation of Smad1 and its Transcriptional Activity

(A) Polyubiquitinylation of Smad1 requires GSK3 and MAPK phosphorylation sites. Cells were co-transfected with Smad1-flag, Smurf1 and Ubiquitin-HA. (B) GSK3 inhibitors decrease polyubiquitinylation of human Smad1. SB415286 (SB4, Biomol) was used at 40 μM, and LiCl at 30 mM, for 24 hours. (C) SGM and SMM are more active than SWT mRNA in a BRE-luciferase transcriptional reporter assay in ectodermal Xenopus explants. (D) The GSK3 inhibitor LiCl increases BMP4-induced transcriptional activation of BRE-luciferase in 293T cells 8 hrs after co-transfection. Data are represented as mean ± standard deviation, three independent experiments.

Figure 3. The Duration of the pSmad1^Cter Signal Is Regulated by MAPK and GSK3

(A) Sequential phosphorylation of Smad1 by BMPR, MAPK and GSK3 after a pulse of BMP7. (B) Induction of pSmad1^MAPK by BMP7 is blocked by triple inhibition of MAPKs (lane 7). Inhibitors used were: MEK/Erk (10 μM U0126), p38 (10 μM Calbiochem CFPD p38 inhibitor), and JNK (25 μM SP600125). Lane 6 contained both p38 and JNK inhibitors. (C) The duration of pSmad1^Cter signal is prolonged by inhibition of MAPKs, GSK3 (40 μM SB415286 or 30 mM LiCl), or proteasome activity (50 μM Lactacystin).

Figure 4. MAPK and GSK3 Phosphorylations Target Smad1 and Total Polyubiquitinated Proteins to the Centrosome in Cos7 Cells

(A–C) pSmad1^Cter accumulates in the centrosome (arrows) in response to proteasomal inhibition by Lactacystin (24%, n=323). (D) The proteasomal subunit S20α5 normally accumulates in the centrosome in Cos7 cells (arrows). (E and F) Nuclear and centrosomal localization of pSmad1^MAPK. (G–I) pSmad1^GSK3- A (green) colocalizes with the centrosome marker γ-Tubulin (red). Note that in panels E through I BMP4 was not added, but the medium contained 10% fetal calf serum, which provides growth factors such as BMP and FGF. (J and K) Specificity of pSmad1^MAPK and pSmad1^GSK3-A staining: phospho-peptide competition and λ-phosphatase sensitivity. (L) pSmad1^GSK3 accumulates in the centrosome 8 hrs after Lactacystin (50 μM) treatment (a 25-fold increase in volume). (M–O) Treatment of Cos7 cells with Lactacystin induces accumulation of total cellular polyubiquitinylated proteins in the centrosomal region marked by Pericentrin in red. In one cell a ring of pericentrosomal staining is seen.

Figure 5. Wnt3a Regulates Smad1 Phosphorylation

(A) Model of biochemical and cellular signaling pathway integration at the level of Smad1 phosphorylations. (B) Wnt3a protein (300 ng/ml, R&D Systems) inhibits Smad1 phosphorylation by GSK3 and stabilizes pSmad1^Cter. L-cells cultured in the absence of serum were treated with purified Wnt3a protein for 1 hour; 0.3 nM BMP7 and 40 ng/ml FGF2 were added after 20 minutes of Wnt treatment. (C) Wnt3a (300 ng/ml) stabilizes pSmad1^Cter and β-Catenin in hESCs after two hours. (D–E) Activation of the Wnt pathway using transfected CA-LRP6 (Tamai et al., 2004) disperses pSmad1^GSK3 from the centrosome into small cytoplasmic puncta. Note that in CA-LRP6 transfected cells the levels of pSmad1^GSK3 in the nucleus are elevated. (F–G) The cytoplasmic puncta containing pSmad1^GSK3 most likely correspond to LRP6-signalosomes because they co-stain with anti-GSK3 antibodies. Transfected cells were identified by co-transfection of CFP, not shown here.

Figure 6. The Dorsalizing Effects of Wnt Inhibition Are Rescued by SGM

(A–C) Dkk1 (n=28) and Chordin (n=45) protein (R&D) injections into the blastula cavity (60 nl of 2.5 μM and 0.5 μM, respectively) present considerable phenotypical similarities. (D) Proposed molecular pathway. (E–H) Dkk1 mRNA suppresses the anti-neural phenotype of BMP4 overexpression (n for each sample was 18, 13, 23 and 18, respectively). (I–P) Wnt8 MO causes dorsalization, which is blocked by GSK3-resistant SGM, but not by SWT mRNA (n for each sample was 22, 17, 23 and 26, respectively). (Q–T) The expansion of the neural plate by Dkk1 mRNA is rescued by GSK3-resistant SGM, but not SWT mRNA (n=30, 22, 21 and 27, respectively).

Figure 7. Wnt Signaling Induces Epidermis in a Smad1/5/8 and β–Catenin Dependent, but Tcf3 Independent, Manner

(A) LiCl induces epidermis (Cytokeratin) and inhibits neural differentiation (NCAM, Otx2). Radioactive RT-PCR analysis of whole embryos (WE), animal cap (AC) explants and dissociated animal cap cells at stage 13. MyoD indicates lack of mesoderm induction and Ornithine decarboxylase (ODC) equal loading. (B) Quantitative PCR of dissociated animal caps injected with pCSKA-Wnt8 and pCS2-LRP6 DNA. DN-Smad5 mRNA was co-injected to block Smad1/5/8 activity. Cytokeratin mRNA levels at stage 13 were normalized for ODC mRNA and the standard deviation from three independent experiments is indicated. (C,C′) Wnt3a protein (60 nl of 16 ng/μl) microinjected into the blastula cavity at stage 9 inhibits anterior neural plate and expands epidermis (n=40 and 42, respectively). (D,D′) DN-Smad5 converts the entire ectoderm into neural tissue and is epistatic to Wnt3a protein injection (n=27 and 35). ADMP MO was co-injected to eliminate all traces of epidermis. (E,E′) Dkk1 mRNA expands the neural plate (n=100 and 53). (F) ΔN-Tcf3 mRNA eliminates the neural plate; only a ring of Sox2 expression in ventral mesoderm remained (90%, n=30). (F′) Dkk1 mRNA rescues neural plate in the presence of ΔN-Tcf3 in 60% of embryos (n=70). (G,G′) The induction of neural plate by Dkk1 mRNA has a complete requirement for β-Catenin (100%, n=17 and 55, respectively). (H) Model in which the BMP (D–V) and Wnt (A–P) patterning pathways are integrated at the level of Smad1/5/8 phosphorylations. Black arrows indicate direct protein-protein interactions and blue arrows transcriptional regulation by Smad1/5/8; all interactions are supported by overexpression or morpholino studies in Xenopus (Lee et al., 2006, and data not shown).