mouse control embryo E17.5

mouse control embryo analyzed at embryonic day E17.

Shh hypomorph mouse embryo E17.5

Shh hypomorph mouse embryo analyzed at embryonic day E17.

A proposed model of RTK and WNT/β-catenin signaling cross-talk.

<p>In the basal cell state, cytoplasmic β-catenin levels are low due to rapid turnover mediated by the destruction complex (AXIN1, APC, CK1 and GSK3), where CK1 and GSK3-mediated phosphorylation targets β-catenin for degradation. WNT binds to its cell surface receptors FRIZZLED and LRP6, inducing the clustering of WNT/FRIZZLED/LRP6 into the multimeric complexes called signalosomes. In signalosomes, LRP6 becomes phosphorylated at PPPS/TP motifs, which allows for AXIN1 and GSK3 binding. Signalosomes also facilitate the amplification of the WNT signal, where initially phosphorylated LRP6 molecules serve as high affinity docking sites for GSK3 that, in turn, phosphorylates additional LRP6 molecules to create even more AXIN1/GSK3 binding sites. AXIN1/GSK3 sequestration by LRP6 leads to dissolution of the destruction complex, allowing for β-catenin stabilization, its nuclear translocation, and activation of gene transcription dependent on TCF/LEF transcription factors. WNT-induced LRP6 phosphorylation requires signalosome assembly and therefore can only involve the mature, transmembrane LRP6. This contrasts with the ERK-mediated LRP6 phosphorylation, since ERK, activated by RTKs, is a cytosolic kinase than can phosphorylate both the mature (transmembrane) LRP6 and immature (intracellular) LRP6 during its Golgi-based membrane transport (gray arrow). In the absence of a signalosome, ERK-phosphorylated LRP6 may recruit a limited amount of AXIN1/GSK3 that is not sufficient to fully stabilize β-catenin. In the presence of WNT, however, LRP6 molecules pre-phosphorylated by ERK integrate into the newly formed signalosomes and help to amplify the WNT signal by providing more initial binding sites for AXIN1/GSK3. In addition to the ERK/LRP6 pathway, RTKs also directly phosphorylate β-catenin at Tyr142, possibly liberating β-catenin from its association with the cell membrane, and allowing for its transcriptional activation.</p

FGF2 uses ERK MAP kinase to phosphorylate LRP6.

<p>(A) RCS cells were treated as indicated and analyzed for activating phosphorylation of JNK, ERK and p38 MAP kinases by WB (C1, C2 - untreated controls). Anizomycin (An.; 10 µg/ml, 1.5 hour) serves as positive control for JNK and p38 activation. (B) Cells were treated with the MEK inhibitor U0126 or FGFR inhibitor SU5402 for 30 minutes prior to FGF2 treatment and analyzed for the indicated molecules. (C) Cells were transfected with Topflash reporter vectors, treated with the U0126 (15 µM) and FGFR inhibitor SU5402 (7 µM) for 1 hour prior to FGF2 and WNT3a addition, and analyzed for luciferase activity. A logarithmic scale for the <i>y</i>-axis is necessary to show the massive Topflash activation induced by FGF2/WNT3a. The data represent an average from three transfections (each measured twice), with the indicated standard deviations (* <i>p</i><0.001; Student’s <i>t</i>-test). Results are representative of three experiments. (D) Active ERK was immunoprecipitated (IP) from cells treated with FGF2 (left panel), and subjected to a kinase assay with either recombinant ELK1 or LRP6 as a substrate (right panel). A sample with ATP omitted serves as a negative control.</p

Col2a1-CreERT2/R26DTA mouse embryo E17.5

Col2a1-CreERT2/R26DTA mouse embryo injected with 2.5 mg tamoxifen at E12.5 and analysed at embryonic day E17.

Disease-associated FGFR3 and FGFR2 mutants signal via ERK/LRP6 pathway.

<p>(A) RCS cells were transfected with wt FGFR3 or activating FGFR3 mutants (N540K, G380R, R248C, Y373C, K650M, K650E), and analyzed for the indicated molecules by WB 48 hours later. The levels of ERK phosphorylation vary among the tested mutants, reflecting the different strength of FGFR3 activation by each particular mutation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035826#pone.0035826-Krejci2" target="_blank">[24]</a>. K508M - kinase inactive FGFR3 mutant. GFP and empty vectors serve as transfection controls. (B) LRP6 phosphorylation at Thr1572 caused by highly activating FGFR3 mutants R248C and K650E. (C) Cells were transfected with the indicated FGFR3 vectors together with Topflash reporter vectors, treated with WNT3a and analyzed for luciferase activity. Data represent an average from three transfections (each measured twice), with the indicated standard deviations. A logarithmic scale of the <i>y</i>-axis is necessary to express the massive Topflash activation in WNT3a-treated cells expressing activating FGFR3 mutants (* <i>p</i><0.001; Student’s <i>t</i>-test; compared to wt FGFR3). Results are representative of four experiments. (D) Cells were transfected with wt FGFR2 or activating FGFR2 mutants (S252W, P253R, C342R, C342Y, Y375C), and analyzed for the indicated molecules by WB. Note the significant ERK and LRP6 phosphorylation caused by C342R, C342Y and Y375C mutants, which correlates with increased basal (E; upper graph) and WNT3a-induced (E; lower graph) β-catenin activity, evidenced by Topflash experiment. Results are representative for three experiments (* <i>p</i><0.001; Student’s <i>t</i>-test; compared to wt FGFR2).</p

EGFR and TRKA activate WNT/β-catenin signaling via ERK/LRP6 pathway.

<p>(A, E) RCS cells were transfected with empty plasmid or plasmid encoding V5-tagged EGFR or TRKA, treated with EGF or NGF (50 ng/ml) for 1 hour, and analyzed for indicated molecules by WB. (B, F) Cells were transfected as indicated, grown for 24 hours, treated with EGF, NGF and WNT3a, and analyzed for luciferase activity. Data represent an average from three transfections (each measured twice). Statistically significant differences are indicated (* <i>p</i><0.0001, # <i>p</i><0.05; Student’s <i>t</i>-test). Note the potent upregulation of basal or WNT3a-mediated Topflash activity in EGF or NGF-treated cells expressing the corresponding receptor. (C, G) Cells were transfected with EGFR (C) or TRKA (G) together with Topflash reporter vectors, treated with U0126 (20 µM) one hour prior to EGF, NGF and WNT3a treatment, and analyzed for luciferase activity. Data represent an average from three or four transfections (each measured twice). Statistically significant differences are indicated (* <i>p</i><0.0001, # <i>p</i><0.005; Student’s <i>t</i>-test, compared to cells without U0126 for each treatment). (D, H) Cells were transfected as indicated, treated with EGF, NGF and WNT3a for 48 hours, and analyzed for luciferase activity. Data represent an average from four transfections (each measured twice), with the indicated standard deviations. Statistically significant differences are indicated (* <i>p</i><0.0001, # <i>p</i><0.001; Student’s <i>t</i>-test).</p

Six1, Six4, double knockout E18.5

Six1, Six4, double knockout analyzed at embryonic day E18.

Nkx2.2-Cre/Shhfloxed/floxed embryos E12.5

Nkx2.2-Cre/Shhfloxed/floxed mouse embryo analyzed at embryonic day E12.

Heterozygous (DEL-90-C2) embryo E17.5

Heterozygous deletion of the genomic region (DEL-90-C2) analyzed at embryonic day E17.