Receptor Tyrosine Kinases Activate Canonical WNT/β-Catenin Signaling via MAP Kinase/LRP6 Pathway and Direct β-Catenin Phosphorylation

Receptor tyrosine kinase signaling cooperates with WNT/β-catenin signaling in regulating many biological processes, but the mechanisms of their interaction remain poorly defined. We describe a potent activation of WNT/β-catenin by FGFR2, FGFR3, EGFR and TRKA kinases, which is independent of the PI3K/AKT pathway. Instead, this phenotype depends on ERK MAP kinase-mediated phosphorylation of WNT co-receptor LRP6 at Ser1490 and Thr1572 during its Golgi network-based maturation process. This phosphorylation dramatically increases the cellular response to WNT. Moreover, FGFR2, FGFR3, EGFR and TRKA directly phosphorylate β-catenin at Tyr142, which is known to increase cytoplasmic β-catenin concentration via release of β-catenin from membranous cadherin complexes. We conclude that signaling via ERK/LRP6 pathway and direct β-catenin phosphorylation at Tyr142 represent two mechanisms used by various receptor tyrosine kinase systems to activate canonical WNT signaling

NF449 Is a Novel Inhibitor of Fibroblast Growth Factor Receptor 3 (FGFR3) Signaling Active in Chondrocytes and Multiple Myeloma Cells*

The FGFR3 receptor tyrosine kinase represents an attractive target for therapy due to its role in several human disorders, including skeletal dysplasias, multiple myeloma, and cervical and bladder carcinomas. By using molecular library screening, we identified a compound named NF449 with inhibitory activity toward FGFR3 signaling. In cultured chondrocytes and murine limb organ culture, NF449 rescued FGFR3-mediated extracellular matrix loss and growth inhibition, which represent two major cellular phenotypes of aberrant FGFR3 signaling in cartilage. Similarly, NF449 antagonized FGFR3 action in the multiple myeloma cell lines OPM2 and KMS11, as evidenced by NF449-mediated reversal of ERK MAPK activation and transcript accumulation of CCL3 and CCL4 chemokines, both of which are induced by FGFR3 activation. In cell-free kinase assays, NF449 inhibited the kinase activity of both wild type and a disease-associated FGFR3 mutant (K650E) in a fashion that appeared non-competitive with ATP. Our data identify NF449 as a novel antagonist of FGFR3 signaling, useful for FGFR3 inhibition alone or in combination with inhibitors that target the ATP binding site

Receptor Tyrosine Kinases Activate Canonical WNT/?-Catenin Signaling via MAP Kinase/LRP6 Pathway and Direct Beta-Catenin Phosphorylation

Receptor tyrosine kinase signaling cooperates with WNT/β-catenin signaling in regulating many biological processes, but the mechanisms of their interaction remain poorly defined. We describe a potent activation of WNT/β-catenin by FGFR2, FGFR3, EGFR and TRKA kinases, which is independent of the PI3K/AKT pathway. Instead, this phenotype depends on ERK MAP kinase-mediated phosphorylation of WNT co-receptor LRP6 at Ser1490 and Thr1572 during its Golgi network-based maturation process. This phosphorylation dramatically increases the cellular response to WNT. Moreover, FGFR2, FGFR3, EGFR and TRKA directly phosphorylate β-catenin at Tyr142, which is known to increase cytoplasmic β-catenin concentration via release of β-catenin from membranous cadherin complexes. We conclude that signaling via ERK/LRP6 pathway and direct β-catenin phosphorylation at Tyr142 represent two mechanisms used by various receptor tyrosine kinase systems to activate canonical WNT signaling

Receptor Tyrosine Kinases Activate Canonical WNT/?-Catenin Signaling via MAP Kinase/LRP6 Pathway and Direct Beta-Catenin Phosphorylation

Receptor tyrosine kinase signaling cooperates with WNT/β-catenin signaling in regulating many biological processes, but the mechanisms of their interaction remain poorly defined. We describe a potent activation of WNT/β-catenin by FGFR2, FGFR3, EGFR and TRKA kinases, which is independent of the PI3K/AKT pathway. Instead, this phenotype depends on ERK MAP kinase-mediated phosphorylation of WNT co-receptor LRP6 at Ser1490 and Thr1572 during its Golgi network-based maturation process. This phosphorylation dramatically increases the cellular response to WNT. Moreover, FGFR2, FGFR3, EGFR and TRKA directly phosphorylate β-catenin at Tyr142, which is known to increase cytoplasmic β-catenin concentration via release of β-catenin from membranous cadherin complexes. We conclude that signaling via ERK/LRP6 pathway and direct β-catenin phosphorylation at Tyr142 represent two mechanisms used by various receptor tyrosine kinase systems to activate canonical WNT signaling

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

RTKs phosphorylate β-catenin at Tyr142.

<p>(A) RCS cells were treated for indicated times with FGF2 (10 ng/ml) in the presence of heparin (1 µg/ml), and analyzed for β-catenin phosphorylation at Tyr142 by WB (arrow). (B) HEK293 cells were transfected with wt FGFR2 or its activating mutant Y375C, and analyzed for indicated molecules 48 hours later. Note the increased β-catenin phosphorylation at Tyr142 (arrow). (C) Active recombinant FGFR3, FGFR2, TRKA and EGFR were subjected to a cell-free kinase assay with recombinant β-catenin as a substrate. Samples with ATP or kinase omitted serve as controls for kinase reaction.</p

ERK phosphorylates LRP6 along its Golgi-based maturation pathway.

<p>(A) The LRP6 phosphorylation induced in RCS cells by FGF2 and WNT3a was determined by WB and quantified by densitometry. LRP6 migrates as two bands, differently phosphorylated in cells treated by FGF2 and WNT3a (arrows). (B, C) Addition of recombinant DICKKOPF1 (Dkk1) prevents WNT3a-mediated LRP6 phosphorylation (B; upper LRP6 band) and Topflash activation (C), but not that induced by FGF2. (D) Inhibition of ERK pathway by U0126 (10 µM) prevents FGF2-mediated LRP6 phosphorylation (both bands; arrows) while it does not affect WNT3a-mediated phosphorylation (upper band). (E) RCS cells were transfected with V5-tagged LRP6 or empty plasmid, incubated in the presence of tunicamycin (0.1 µM) for 24 hours, and analyzed for indicated molecules by WB. (F) HEK293 cells were transfected with V5-tagged LRP6 plasmid and treated with brefeldin A for 24 hours. Empty vector (pcDNA3) serves as a control for transfection; ACTIN or α-TUBULIN serve as loading controls. LRP6 fails to fully mature in cells treated with tunicamycin or brefeldin A (arrows), which inhibit glycosylation or protein transport from the endoplasmatic reticulum to the Golgi network, respectively. (G) A whole RCS cell lysate was incubated with N-glycosidase F (PNG-F) for 16 hours and analyzed for LRP6 by WB. (H) RCS cells were cultivated with tunicamycin (5 µM) for 24 hours prior to FGF2 and WNT3a (1 hour) treatment. Note the lack of WNT3a-mediated LRP6 phosphorylation when only the immature, non-glycosylated LRP6 is produced as a result of tunicamycin treatment.</p

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