November 30, 2007;
Integrating patterning signals: Wnt/GSK3 regulates the duration of the BMP/Smad1 signal.
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
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
, 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.
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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 Ser214 (pSmad1MAPK) and Ser210 (pSmad1GSK3 antibody B was used).(N) Phospho-specific antibodies (pSmad1MAPK and pSmad1GSK3-A) demonstrate that GSK3 phosphorylation of recombinant Smad1 requires MAPK priming, in nonradioactive Western blots. Recombinant Smad1 substrate was used in the same amount as in (K).
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 antineural 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 coinjected 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 and 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 and D′) DN-Smad5 converts the entire ectoderm into neural tissue and is epistatic to Wnt3a protein injection (n = 27 and 35). ADMP MO was coinjected to eliminate all traces of epidermis.(E and 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 and 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).
Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation.