Regulation of Lymphoid Enhancer Factor 1/T-Cell Factor by Mitogen-Activated Protein Kinase-Related Nemo-Like Kinase-Dependent Phosphorylation in Wnt/β-Catenin Signaling

WRM-1 is only 19% identical to vertebrate β-catenin.

“Indeed, WRM-1, which is only 19% identical to vertebrate β-catenin, does not bind to the POP-1 β-catenin-binding domain per se but weakly binds full-length POP-1 (14, 19, 22).”

• cites [14] sourceNot checked — no full text of the source
• cites [19] sourceDisputed

  The percentage-identity table in the section on β-catenin family members across species (page unavailable) gives 17% identity between WRM-1 and human β-catenin, not 19%. The Methods section says these percentages were calculated from sequence alignments. (LLM, conf=0.90)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [22] sourceNot checked — no full text of the source

HeLa cells contain low levels of endogenous LEF-1/TCF.

“HeLa cells, which contain low levels of endogenous LEF-1/TCF (10).”

• cites [10] sourcePartly supported

  The Results (page not provided) describe HeLa cells as not expressing LEF-1, supporting low endogenous LEF-1 relative to LEF-1-expressing Jurkat cells. The passage does not establish the level of endogenous TCF in HeLa cells. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

WRM-1 does not bind to the POP-1 β-catenin-binding domain per se.

“Indeed, WRM-1, which is only 19% identical to vertebrate β-catenin, does not bind to the POP-1 β-catenin-binding domain per se but weakly binds full-length POP-1 (14, 19, 22).”

• cites [14] sourceNot checked — no full text of the source
• cites [19] sourcePartly supported

  In the protein-interaction results (page unavailable), WRM-1 interacts weakly with POP-1 even after deletion of POP-1’s amino-terminal β-catenin-binding region. This supports an interaction that does not require that region, but the paper does not test whether WRM-1 can bind the region itself; it presents a different mode of interaction as a suggestion, not a categorical finding. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [22] sourceNot checked — no full text of the source

WRM-1 weakly binds full-length POP-1.

“Indeed, WRM-1, which is only 19% identical to vertebrate β-catenin, does not bind to the POP-1 β-catenin-binding domain per se but weakly binds full-length POP-1 (14, 19, 22).”

• cites [14] sourceNot checked — no full text of the source
• cites [19] sourceSupported
• cites [22] sourceNot checked — no full text of the source

NLK phosphorylates LEF-1/TCF on two serine/threonine residues located in its central region.

“NLK phosphorylates LEF-1/TCF on two serine/threonine residues located in its central region.”

Mutation of both phosphorylated residues of LEF-1/TCF to alanine rendered LEF-1 resistant to inhibition by NLK.

“Mutation of both residues to alanine enhanced LEF-1 transcriptional activity and rendered it resistant to inhibition by NLK.”

Mutation of both phosphorylated residues of LEF-1/TCF to alanine enhanced LEF-1 transcriptional activity.

“Mutation of both residues to alanine enhanced LEF-1 transcriptional activity and rendered it resistant to inhibition by NLK.”

NLK interacts indirectly with β-catenin via LEF-1/TCF to form a complex.

“We found that NLK interacts directly with LEF-1/TCF and indirectly with β-catenin via LEF-1/TCF to form a complex.”

NLK interacts directly with LEF-1/TCF.

“We found that NLK interacts directly with LEF-1/TCF and indirectly with β-catenin via LEF-1/TCF to form a complex.”

Phosphorylation of TCF-4 by NLK inhibited DNA binding by the β-catenin-TCF-4 complex.

“Phosphorylation of TCF-4 by NLK inhibited DNA binding by the β-catenin-TCF-4 complex.”

The inhibition of DNA binding by the β-catenin-TCF-4 complex was abrogated when a mutant form of TCF-4 was used in which both threonines were replaced with valines.

“However, this inhibition was abrogated when a mutant form of TCF-4 was used in which both threonines were replaced with valines.”

Phosphorylation of TCF-4(2V) by NLK is greatly decreased in comparison with that of wild-type TCF-4.

“In vitro kinase reactions showed that phosphorylation of TCF-4(2V) by NLK was greatly decreased in comparison with that of wild-type TCF-4 (Fig. 4C, top panel, lanes 1 and 3).”

Wnt-1 stimulation induced expression of the TOPFLASH reporter about fourfold over vector-transfected cells.

“Stimulation with Wnt-1 induced expression of this reporter gene about fourfold over that of the vector-transfected cells (Fig. 6, lanes 1 and 4).”

No activity was observed when a FOPFLASH reporter lacking LEF-1/TCF-binding sites was cotransfected.

“In contrast, no activity was observed when a FOPFLASH reporter, which lacks LEF-1/TCF-binding sites, was cotransfected (Fig. 6, lane 14).”

Flag-NLK and T7-LEF-1ΔN interact when coexpressed.

“We observed that Flag-NLK and T7-LEF-1ΔN could interact when coexpressed (Fig. 1, top panel, lane 8), indicating that the NH2-terminal deletion of LEF-1 does not affect its association with NLK.”

LEF-1 associates with NLK.

“LEF-1 was found to associate with NLK (Fig. 1, top panel, lane 6).”

Coexpression of kinase-negative NLK(K155M) did not inhibit reporter activity.

“whereas coexpression of the kinase-negative mutant form NLK(K155M) did not (Fig. 6, lane 8).”

NLK associates with both LEF-1ΔN and LEF-1ΔC.

“In this assay, NLK was found to associate with both LEF-1ΔN and LEF-1ΔC (Fig. 3, top panel, lanes 5 and 6).”

• AssumptionRests on an assumption: The LEF-1 protein contains a COOH-terminal HMG DNA-binding domain and an NH2-terminal β-catenin-binding domain.

In the absence of exogenous TCF-4, little β-catenin is detected in NLK immunocomplexes precipitated with anti-Flag antibody.

“We observed that, in the absence of exogenous TCF-4, little β-catenin was detected in the NLK immunocomplexes precipitated with the anti-Flag antibody (Fig. 2, second panel, lane 3).”

NLK phosphorylation of LEF-1ΔN and LEF-1ΔC is dependent on the kinase activity of NLK.

“As shown in Fig. 3B, NLK phosphorylated both LEF-1ΔN and LEF-1ΔC in vitro (Fig. 3, bottom panel, lanes 5 and 6) and this phosphorylation was dependent on the kinase activity of NLK (Fig. 3, bottom panel, lanes 8 and 9).”

• AssumptionRests on an assumption: The LEF-1 protein contains a COOH-terminal HMG DNA-binding domain and an NH2-terminal β-catenin-binding domain.

Coexpression of LEF-1 dramatically increases the interaction between β-catenin and NLK.

“We observed that coexpression of LEF-1 dramatically increased the interaction between β-catenin and NLK (Fig. 1, middle panel, lane 7).”

Two NLK phosphorylation sites exist in LEF-1/TCF.

“We identify two NLK phosphorylation sites in LEF-1/TCF and present data suggesting that phosphorylation of these sites contributes to the down-regulation of LEF-1/TCF transcriptional activity.”

When TCF-4 is coexpressed, the amount of β-catenin detected in NLK immunocomplexes increases.

“However, when TCF-4 was coexpressed, the amount of β-catenin detected in the immunocomplexes increased (Fig. 2, second panel, lane 5).”

Expression of mutant LEF-1(2A) alone did not activate the reporter.

“Expression of the mutant form LEF-1(2A) alone did not activate the reporter (Fig. 6, lane 3)”

Wild-type LEF-1 is efficiently phosphorylated by active but not inactive NLK.

“Wild-type LEF-1 was efficiently phosphorylated by active but not inactive NLK (Fig. 4B, top panel, lanes 1 and 2).”

The mutated LEF-1(2A) protein is only weakly phosphorylated by NLK.

“On the other hand, the mutated LEF-1(2A) protein was only weakly phosphorylated by NLK (Fig. 4B, top panel, lane 3).”

Coexpression of NLK with Wnt-1 and LEF-1 inhibited reporter activity.

“When NLK was coexpressed with Wnt-1 and LEF-1, it inhibited reporter activity (Fig. 6, lane 7)”

NLK association with LEF-1(2A) and TCF-4(2V) is normal.

“This analysis revealed that NLK association with LEF-1(2A) (Fig. 5, top panel, lane 4) and TCF-4(2V) (Fig. 5, top panel, lane 8) was normal”

LEF-1 and TCF-4 proteins migrate more slowly on SDS-PAGE when wild-type NLK is coexpressed.

“Western blot analysis revealed that the LEF-1 and TCF-4 proteins migrated more slowly on SDS-PAGE when wild-type NLK was coexpressed (Fig. 4D, lanes 2 and 7).”

NLK phosphorylates both LEF-1ΔN and LEF-1ΔC in vitro.

“As shown in Fig. 3B, NLK phosphorylated both LEF-1ΔN and LEF-1ΔC in vitro (Fig. 3, bottom panel, lanes 5 and 6) and this phosphorylation was dependent on the kinase activity of NLK (Fig. 3, bottom panel, lanes 8 and 9).”

• AssumptionRests on an assumption: The LEF-1 protein contains a COOH-terminal HMG DNA-binding domain and an NH2-terminal β-catenin-binding domain.

NLK phosphorylates LEF-1/TCF.

“In the present report, we show that NLK directly interacts with and phosphorylates LEF-1/TCF.”

Expression of the kinase-negative mutant NLK(K155M) fails to cause mobility shifts of LEF-1 and TCF-4.

“Expression of the kinase-negative mutant NLK(K155M) failed to cause mobility shifts of LEF-1 (lane 3) and TCF-4 (lane 8).”

When LEF-1(2A) or TCF-4(2V) is coexpressed along with NLK, a fractional shift is observed.

“Furthermore, when LEF-1(2A) or TCF-4(2V) was coexpressed along with NLK, a fractional shift was observed (lanes 5 and 10)”

NLK association with β-catenin is barely detectable.

“In contrast, NLK association with β-catenin was barely detectable (Fig. 1, middle panel, lane 5).”

The mutated forms of LEF-1 and TCF-4 associate with β-catenin to the same extent as each of the respective wild-type forms.

“We found that the mutated forms of LEF-1 and TCF-4 associated with β-catenin to the same extent as each of the respective wild-type forms”

Cotransfection of β-catenin enhances NLK phosphorylation of TCF-4.

“However, cotransfection of β-catenin enhanced NLK phosphorylation of TCF-4 (Fig. 2, bottom panel, lane 5).”

Coexpression of LEF-1(2A) with Wnt-1 stimulated Wnt-1-induced reporter activity more strongly than did expression of wild-type LEF-1.

“but coexpression of LEF-1(2A) with Wnt-1 stimulated Wnt-1-induced reporter activity more strongly than did expression of wild-type LEF-1 (Fig. 6, lane 9).”

A kinase-negative form of NLK, NLK(K155M), interacts with LEF-1, LEF-1ΔN, and LEF-1ΔC.

“A kinase-negative form of NLK, NLK(K155M), also interacted with LEF-1, LEF-1ΔN, and LEF-1ΔC (Fig. 3, top panel, lanes 7 to 9).”

Examination of the primary sequences of the LEF-1/TCF middle regions revealed two potential NLK phosphorylation sites that are conserved among human LEF-1, human TCF-4, and C. elegans POP-1.

“Examination of the primary sequences of the LEF-1/TCF middle regions revealed two potential NLK phosphorylation sites that are conserved among human LEF-1, human TCF-4, and C. elegans POP-1 (Fig. 4A).”

• AssumptionRests on an assumption: The LEF-1 protein contains a COOH-terminal HMG DNA-binding domain and an NH2-terminal β-catenin-binding domain.

Precipitation of LEF-1(2A) with NLK is normal.

“Precipitation of LEF-1(2A) with NLK was normal (Fig. 4B, middle panel, lane 3)”

LEF-1 was not observed to cause enhancement of NLK phosphorylation of TCF-4.

“In a parallel experiment, LEF-1 was not observed to cause such enhancement (data not shown).”

Coexpression of LEF-1 and Wnt-1 enhanced Wnt-1-induced activation of the reporter.

“However, coexpression of LEF-1 and Wnt-1 enhanced Wnt-1-induced activation of the reporter (Fig. 6, lane 6).”

Flag-NLK and TCF-4 interact when coexpressed in 293 cells.

“We confirmed this interaction by coexpressing Flag-NLK and TCF-4 in 293 cells (Fig. 2, top panel, lane 4).”

The binding of NLK to LEF-1 is detected by yeast two-hybrid.

“The binding of NLK to LEF-1 was also detected by using a yeast two-hybrid system (data not shown), supporting the idea that the interaction between these two proteins is direct.”

Coexpression of LEF-1ΔN does not enhance the association between NLK and β-catenin.

“However, coexpression of LEF-1ΔN did not enhance the association between NLK and β-catenin (Fig. 1, middle panel, lane 9), as had been observed with wild-type LEF-1.”

Expression of LEF-1 alone did not augment activation of the reporter gene.

“Expression of LEF-1 alone did not augment the activation of the reporter gene (lane 2).”

NLK phosphorylates TCF-4 in vitro even in the absence of β-catenin transfection.

“As observed previously (13), NLK phosphorylated TCF-4 in vitro even in the absence of β-catenin transfection (Fig. 2, bottom panel, lane 4).”

• cites [13] sourceNot checked — no full text of the source

Phosphorylation of TCF-4 by NLK did not affect its interaction with β-catenin in vitro.

“Western blot analysis confirmed that neither mutation of TCF-4 to TCF-4(2V) nor phosphorylation of TCF-4 by NLK affected its interaction with β-catenin in vitro (Fig. 7A).”

NLK did not inhibit reporter activity induced by coexpression of LEF-1(2A) with Wnt-1.

“However, NLK did not inhibit reporter activity induced by coexpression of LEF-1(2A) with Wnt-1 (Fig. 6, lane 10).”

Mutation of TCF-4 to TCF-4(2V) did not affect its interaction with β-catenin in vitro.

“Western blot analysis confirmed that neither mutation of TCF-4 to TCF-4(2V) nor phosphorylation of TCF-4 by NLK affected its interaction with β-catenin in vitro (Fig. 7A).”

The band was supershifted by anti-β-catenin and anti-TCF-4 antibodies, confirming that it contained the β-catenin-TCF-4-DNA complex.

“As expected, this band was supershifted by anti-β-catenin (lane 5) and anti-TCF-4 (lane 9) antibodies, confirming that it contained the β-catenin-TCF-4-DNA complex.”

The NLK phosphorylation sites map to Thr-155 and Ser-166 of LEF-1 and Thr-178 and Thr-189 of TCF-4 in vitro and in vivo.

“Specifically, these sites mapped, in vitro and in vivo, to Thr-155 and Ser-166 of LEF-1 and Thr-178 and Thr-189 of TCF-4.”

When nuclear extracts containing β-catenin were mixed with those from cells coexpressing wild-type TCF-4 and NLK, formation of the β-catenin-TCF-4-DNA complex was greatly reduced.

“In contrast, when nuclear extracts containing β-catenin were mixed with those prepared from cells coexpressing wild-type TCF-4 and NLK, the formation of the β-catenin-TCF-4-DNA complex was greatly reduced (Fig. 7B, lane 3).”

In vertebrates, LEF-1/TCF binds directly to NLK.

“In contrast, in vertebrates, LEF-1/TCF binds directly to NLK and β-catenin interacts indirectly with NLK via LEF-1/TCF to form a larger complex.”

• Also stated in: § DISCUSSION · hedged

  “Our observation that NLK interacts directly with LEF-1/TCF makes it likely that LIT-1 would interact directly with POP-1 in the control of T-cell polarity.”

The mutant TCF-4(2V)-β-catenin complex remained bound to DNA even when NLK was expressed.

“Moreover, the mutant TCF-4(2V)-β-catenin complex remained bound to DNA even when NLK was expressed (Fig. 7B, lane 4)”

The TCF-4(2V) mutation enhanced DNA-binding activity.

“As shown in lane 2, we found that the TCF-4(2V) mutation actually enhanced DNA-binding activity.”

Site-directed mutagenesis of these conserved serine and threonine residues abrogated NLK-mediated phosphorylation in vitro and in vivo.

“Site-directed mutagenesis of these conserved serine and threonine residues abrogated NLK-mediated phosphorylation in vitro and in vivo.”

A specific band was detected when mixtures of nuclear extracts containing wild-type TCF-4 and β-catenin were incubated with a TCF-binding probe.

“A specific band was detected when mixtures of nuclear extracts containing wild-type TCF-4 and β-catenin were incubated with the probe (Fig. 7B, lane 1).”

NLK overexpression failed to suppress endogenous LEF-1/TCF-dependent transcription.

“NLK overexpression failed to suppress the endogenous LEF-1/TCF-dependent transcription (lanes 4 and 5).”

The primary sites of NLK phosphorylation are located in the core region between the β-catenin-binding domain and the DNA-binding domain in the LEF-1 and TCF-4 proteins.

“In this study, we found that the primary sites of NLK phosphorylation are located in the core region between the β-catenin-binding domain and the DNA-binding domain in the LEF-1 and TCF-4 proteins.”

Mutational replacement of these serine and threonine residues with alanine or valine prevents NLK-mediated negative regulation of LEF-1 and TCF-4.

“Mutational replacement of these serine and threonine residues with alanine or valine prevents NLK-mediated negative regulation of LEF-1 and TCF-4.”

The paper identifies two NLK phosphorylation sites in LEF-1 and TCF-4.

“In this report, we identify two NLK phosphorylation sites in LEF-1 and TCF-4”

A MAPK-related pathway acts in concert with Wnt signaling to establish anterior-posterior polarity in C. elegans.

“Thus, a MAPK-related pathway acts in concert with Wnt signaling to establish anterior-posterior polarity in C. elegans.”

• cites [16] sourceNot checked — no full text of the source
• cites [22] sourceNot checked — no full text of the source
• cites [27] Shin T. H., 1999, Cell sourceNot checked — no full text of the source

The lit-1 gene encodes a MAPK-like protein similar to the Drosophila Nemo protein kinase and the mouse Nemo-like kinase (NLK).

“The lit-1 gene encodes a mitogen-activated protein kinase (MAPK)-like protein similar to the Drosophila Nemo protein kinase and the mouse Nemo-like kinase (NLK).”

• cites [16] sourceNot checked — no full text of the source
• cites [22] sourceNot checked — no full text of the source
• cites [27] Shin T. H., 1999, Cell sourceNot checked — no full text of the source

MOM-4 can activate kinase activity of the LIT-1-WRM-1 complex.

“Furthermore, MOM-4 can activate kinase activity of the LIT-1-WRM-1 complex (27).”

• cites [27] Shin T. H., 1999, Cell sourceNot checked — no full text of the source

The mom-4 gene encodes a MAPK kinase kinase-like protein related to the mammalian protein kinase TAK1.

“The mom-4 gene encodes a MAPK kinase kinase-like protein related to the mammalian protein kinase TAK1.”

• cites [16] sourceNot checked — no full text of the source
• cites [22] sourceNot checked — no full text of the source
• cites [27] Shin T. H., 1999, Cell sourceNot checked — no full text of the source

Mutations in lit-1 or mom-4 cause a loss of polarity and POP-1 asymmetry.

“Mutations in lit-1 or mom-4 cause a loss of polarity and POP-1 asymmetry (16, 22, 27).”

• cites [16] sourceNot checked — no full text of the source
• cites [22] sourceNot checked — no full text of the source
• cites [27] Shin T. H., 1999, Cell sourceNot checked — no full text of the source

NLK phosphorylation on these sites contributes to the down-regulation of LEF-1/TCF transcriptional activity.

“These results suggest that NLK phosphorylation on these sites contributes to the down-regulation of LEF-1/TCF transcriptional activity.”

In C. elegans, WRM-1 down-regulates, rather than activates, POP-1.

“whereas in C. elegans, WRM-1 down-regulates, rather than activates, POP-1 (16, 21, 22, 30).”

• cites [16] sourceNot checked — no full text of the source
• cites [21] sourceNot checked — no full text of the source
• cites [22] sourceNot checked — no full text of the source

The Wnt signaling pathway in mammalian cells is regulated by a MAPK-related pathway composed of TAK1 and NLK.

“Recent evidence indicates that the Wnt signaling pathway in mammalian cells is also regulated by a MAPK-related pathway composed of MAPK kinase kinase TAK1 and MAPK-related NLK (13).”

• cites [13] sourceNot checked — no full text of the source

TAK1 and NLK are negative regulators of LEF-1/TCF activity.

“Thus, TAK1 and NLK are negative regulators of LEF-1/TCF activity.”

A specific MAP kinase pathway involving TAK1 and NLK suppresses Wnt signaling.

“Previously, we have shown that a specific mitogen-activated protein (MAP) kinase pathway involving the MAP kinase kinase kinase TAK1 and MAP kinase-related Nemo-like kinase (NLK) suppresses Wnt signaling.”

β-Catenin is recruited to the NLK-LEF-1 complex via interaction with the NH2 terminus of LEF-1.

“These results suggest that β-catenin is recruited to the NLK-LEF-1 complex via interaction with the NH2 terminus of LEF-1.”

β-Catenin forms a complex with NLK in a LEF-1/TCF-dependent manner.

“β-Catenin forms a complex with NLK in a LEF-1/TCF-dependent manner.”

• Also stated in: § RESULTS · hedged

  “These results suggest that LEF-1 binds directly to NLK, while β-catenin interacts indirectly with NLK via binding to LEF-1.”

The NH2-terminal deletion of LEF-1 does not affect its association with NLK.

“We observed that Flag-NLK and T7-LEF-1ΔN could interact when coexpressed (Fig. 1, top panel, lane 8), indicating that the NH2-terminal deletion of LEF-1 does not affect its association with NLK.”

TAK1-NLK and MOM-4-LIT-1 function analogously to regulate Wnt signaling pathways in mammalian cells and C. elegans, respectively.

“Thus, TAK1-NLK and MOM-4-LIT-1 appear to function analogously to regulate Wnt signaling pathways in mammalian cells and C. elegans, respectively.”

Mutations at the sites of NLK phosphorylation probably do not alter the global three-dimensional structure of LEF-1 or TCF-4.

“indicating that mutations at the sites of NLK phosphorylation probably do not alter the global three-dimensional structure of LEF-1 or TCF-4.”

• AssumptionRests on an assumption: The LEF-1 protein contains a COOH-terminal HMG DNA-binding domain and an NH2-terminal β-catenin-binding domain.

NLK inhibits LEF-1-dependent activation of the LEF-1/TCF reporter.

“This indicates that NLK inhibits LEF-1-dependent activation of the LEF-1/TCF reporter”

NLK does not directly interact with β-catenin.

“NLK directly interacts with LEF-1/TCF but not with β-catenin.”

• Also stated in: § RESULTS · hedged

  “In contrast, NLK association with β-catenin was barely detectable (Fig. 1, middle panel, lane 5).”

NLK phosphorylates LEF-1 and TCF-4 at these serine/threonine residues in vivo.

“These results are consistent with the idea that NLK phosphorylates LEF-1 and TCF-4 at these serine/threonine residues in vivo.”

• AssumptionRests on an assumption: The LEF-1 protein contains a COOH-terminal HMG DNA-binding domain and an NH2-terminal β-catenin-binding domain.

The LEF-1(2A) mutation does not affect its interaction with NLK in vitro.

“suggesting that the LEF-1(2A) mutation does not affect its interaction with NLK in vitro.”

The NLK phosphorylation sites are located in the middle region between the β-catenin-binding domain and the DNA-binding domain of LEF-1.

“These results suggest that the NLK phosphorylation sites are also located in the middle region between the β-catenin-binding domain and the DNA-binding domain of LEF-1.”

• AssumptionRests on an assumption: The LEF-1 protein contains a COOH-terminal HMG DNA-binding domain and an NH2-terminal β-catenin-binding domain.

NLK interacts with the β-catenin-LEF-1/TCF complex.

“We have previously shown that NLK interacts with the β-catenin-LEF-1/TCF complex (13).”

• cites [13] sourceNot checked — no full text of the source

A small deletion removing the NH2-terminal 62 amino acids of LEF-1 (LEF-1ΔN) abrogates its interaction with β-catenin.

“A small deletion removing the NH2-terminal 62 amino acids of LEF-1 (LEF-1ΔN) abrogated its interaction with β-catenin (data not shown) (2).”

• cites [2] sourceNot checked — no full text of the source

The full mobility shift requires phosphorylation of both sites.

“indicating that the full mobility shift requires phosphorylation of both sites.”

• AssumptionRests on an assumption: The LEF-1 protein contains a COOH-terminal HMG DNA-binding domain and an NH2-terminal β-catenin-binding domain.

LEF-1/TCF forms a bridge between β-catenin and NLK.

“This supports the possibility that LEF-1/TCF forms a bridge between β-catenin and NLK.”

NLK catalyzes the phosphorylation of Thr-155 and Ser-166 in LEF-1 in vitro.

“These results suggest that NLK catalyzes the phosphorylation of Thr-155 and Ser-166 in LEF-1 in vitro.”

• AssumptionRests on an assumption: The LEF-1 protein contains a COOH-terminal HMG DNA-binding domain and an NH2-terminal β-catenin-binding domain.

NLK associates with TCF-4.

“We have previously shown that NLK associates with TCF-4 (13).”

• cites [13] sourceNot checked — no full text of the source

The mobility shifts of LEF-1 and TCF-4 on SDS-PAGE are due to phosphorylation.

“These mobility shifts were shown to be due to phosphorylation (13).”

• cites [13] sourceNot checked — no full text of the source

The middle region of LEF-1 contributes to NLK interaction.

“This result indicates that the middle region of LEF-1 contributes to NLK interaction.”

• AssumptionRests on an assumption: The LEF-1 protein contains a COOH-terminal HMG DNA-binding domain and an NH2-terminal β-catenin-binding domain.

β-Catenin binds to a region within the NH2-terminal region of LEF-1/TCF.

“β-Catenin binds to a region within the NH2-terminal region of LEF-1/TCF (see Fig. 3A) (2).”

• cites [2] sourceNot checked — no full text of the source

NLK phosphorylates at least the Thr-178 and Thr-189 sites of TCF-4 in vitro.

“This suggests that NLK phosphorylates at least the Thr-178 and Thr-189 sites of TCF-4 in vitro.”

• AssumptionRests on an assumption: The LEF-1 protein contains a COOH-terminal HMG DNA-binding domain and an NH2-terminal β-catenin-binding domain.

Phosphorylation of the two NLK sites in LEF-1/TCF contributes to down-regulation of LEF-1/TCF transcriptional activity.

“We identify two NLK phosphorylation sites in LEF-1/TCF and present data suggesting that phosphorylation of these sites contributes to the down-regulation of LEF-1/TCF transcriptional activity.”

NLK may phosphorylate the β-catenin-TCF-4 complex more efficiently than TCF-4.

“This suggests that NLK may phosphorylate the β-catenin-TCF-4 complex more efficiently than TCF-4.”

LEF-1 and TCF-4 may contain additional NLK phosphorylation sites.

“The fact that some weak phosphorylation was still observed with the LEF-1(2A) and TCF-4(2V) mutant forms suggests that LEF-1 and TCF-4 may contain additional NLK phosphorylation sites.”

• AssumptionRests on an assumption: The LEF-1 protein contains a COOH-terminal HMG DNA-binding domain and an NH2-terminal β-catenin-binding domain.

NLK directly interacts with LEF-1/TCF.

“In the present report, we show that NLK directly interacts with and phosphorylates LEF-1/TCF.”

• Also stated in: § RESULTS · plain

  “NLK directly interacts with LEF-1/TCF but not with β-catenin.”

• Also stated in: § RESULTS · hedged

  “These results suggest that LEF-1 binds directly to NLK, while β-catenin interacts indirectly with NLK via binding to LEF-1.”

TAK1 and NLK act in a pathway parallel to the Wnt pathway.

“These results suggest that TAK1 and NLK act in a pathway parallel to the Wnt pathway.”

Neither WRM-1 nor any other β-catenin homolog is involved in T-cell polarity.

“Although LIT-1 controls both EMS and T-cell polarities, neither WRM-1 nor any other β-catenin homolog is involved in T-cell polarity (9).”

• cites [9] Herman M. C., 2001, Development sourceNot checked — no full text of the source

The C. elegans LIT-1 protein interacts directly with WRM-1.

“The C. elegans LIT-1 protein interacts directly with WRM-1, and this complex can phosphorylate POP-1 in a manner that requires the LIT-1 kinase domain (22).”

• cites [22] sourceNot checked — no full text of the source

NLK phosphorylation of the two sites on wild-type TCF-4 causes decreased DNA binding.

“suggesting that NLK phosphorylation of the two sites on wild-type TCF-4 causes decreased DNA binding.”

LIT-1 controls both EMS and T-cell polarities.

“Although LIT-1 controls both EMS and T-cell polarities, neither WRM-1 nor any other β-catenin homolog is involved in T-cell polarity (9).”

• cites [9] Herman M. C., 2001, Development sourceNot checked — no full text of the source

WRM-1 is required for POP-1 phosphorylation.

“This indicates that WRM-1 is required for POP-1 phosphorylation.”

Phosphorylation of Thr-178 and Thr-189 in TCF-4 mediates the inhibitory effect of NLK.

“These results support a model whereby phosphorylation of Thr-178 and Thr-189 in TCF-4 mediates the inhibitory effect of NLK.”

The inhibition of LEF-1-dependent reporter activation by NLK requires NLK kinase activity.

“and that this inhibition requires NLK kinase activity.”

C. elegans WRM-1 possesses important functional differences from other family members.

“In particular, C. elegans WRM-1 appears to possess important functional differences from other family members.”

• AssumptionRests on an assumption: The components of the Wnt pathway (β-catenin, LEF-1/TCF, NLK) are conserved in vertebrates.

Wnt signaling down-regulates the activity of POP-1.

“POP-1, a LEF-1/TCF-related protein, functions to repress gene expression, and Wnt signaling down-regulates the activity of POP-1 (15, 16, 21, 22, 30).”

• cites [15] sourceNot checked — no full text of the source
• cites [16] sourceNot checked — no full text of the source
• cites [21] sourceNot checked — no full text of the source

HeLa cells may contain an LEF-1/TCF complex that is resistant to NLK-mediated repression.

“This suggests that HeLa cells may contain an LEF-1/TCF complex that is resistant to NLK-mediated repression.”

In vertebrates, β-catenin interacts indirectly with NLK via LEF-1/TCF to form a larger complex.

“In contrast, in vertebrates, LEF-1/TCF binds directly to NLK and β-catenin interacts indirectly with NLK via LEF-1/TCF to form a larger complex.”

The presence of β-catenin promotes phosphorylation of TCF-4 by NLK.

“The presence of β-catenin enhances the interaction between NLK and TCF-4 and thereby promotes phosphorylation of TCF-4 by NLK.”

POP-1 plays a negative role in Wnt signaling.

“POP-1 plays a negative role in Wnt signaling and is inhibited by WRM-1 through LIT-1-mediated phosphorylation of POP-1 (22).”

• cites [22] sourceNot checked — no full text of the source

WRM-1 and POP-1 have opposing functions rather than working as cofactors.

“Thus, WRM-1 and POP-1, rather than working as cofactors, have opposing functions.”

• cites [22] sourceNot checked — no full text of the source

Repression of LEF-1 activity by NLK requires the two phosphorylation sites in LEF-1.

“These results suggest that the repression of LEF-1 activity by NLK requires the two phosphorylation sites in LEF-1.”

NLK phosphorylation of TCF-4 prevents the β-catenin-TCF-4 complex from binding DNA.

“Thus, as observed previously (13), NLK phosphorylation of TCF-4 prevents the β-catenin-TCF-4 complex from binding DNA.”

• cites [13] sourceNot checked — no full text of the source

The presence of β-catenin enhances the interaction between NLK and TCF-4.

“The presence of β-catenin enhances the interaction between NLK and TCF-4 and thereby promotes phosphorylation of TCF-4 by NLK.”

POP-1, a LEF-1/TCF-related protein, functions to repress gene expression.

“POP-1, a LEF-1/TCF-related protein, functions to repress gene expression, and Wnt signaling down-regulates the activity of POP-1 (15, 16, 21, 22, 30).”

• cites [15] sourceNot checked — no full text of the source
• cites [16] sourceNot checked — no full text of the source
• cites [21] sourceNot checked — no full text of the source

NLK expression interferes with the assembly of the β-catenin-TCF-4-DNA complex.

“This indicates that NLK expression interferes with the assembly of the complex.”

It is likely that LIT-1 interacts directly with POP-1 in the control of T-cell polarity.

“Our observation that NLK interacts directly with LEF-1/TCF makes it likely that LIT-1 would interact directly with POP-1 in the control of T-cell polarity.”

• AssumptionRests on an assumption: The components of the Wnt pathway (β-catenin, LEF-1/TCF, NLK) are conserved in vertebrates.

The mechanism for inhibition of the β-catenin-TCF-4 complex DNA binding by NLK-mediated threonine phosphorylation is presumably not caused by direct phosphorylation of the DNA-binding domain, since the sites of NLK phosphorylation are located distal to the DNA-binding domain.

“Although we have not determined the mechanism for inhibition of the β-catenin-TCF-4 complex DNA binding by NLK-mediated threonine phosphorylation, it is presumably not caused by direct phosphorylation of the DNA-binding domain, since the sites of NLK phosphorylation are located distal to the DNA-binding domain.”

• AssumptionRests on an assumption: In many cases in which phosphorylation inhibits DNA binding, the phosphorylation sites are located either within or near the DNA-binding domain.
• AssumptionRests on an assumption: In such cases, phosphorylation interferes with DNA binding by electrostatic repulsion between phosphate groups on the protein and the DNA.

Phosphorylation of these sites contributes to the down-regulation of the ability of LEF-1/TCF transcriptional activity.

“present data suggesting that phosphorylation of these sites contributes to the down-regulation of the ability of LEF-1/TCF transcriptional activity.”

NLK phosphorylation prevents DNA binding of the β-catenin-TCF-4 complex.

“yet it does prevent DNA binding of the β-catenin-TCF-4 complex”

• cites [13] sourceNot checked — no full text of the source

NLK can regulate the transcriptional activity of a β-catenin-LEF-1/TCF complex.

“We have previously demonstrated that NLK can regulate the transcriptional activity of a β-catenin-LEF-1/TCF complex (13).”

• cites [13] sourceNot checked — no full text of the source

NLK phosphorylation does not disrupt the formation of a complex between LEF-1/TCF and β-catenin.

“This phosphorylation does not disrupt the formation of a complex between LEF-1/TCF and β-catenin”

• cites [13] sourceNot checked — no full text of the source

Phosphorylation inhibits LEF-1/TCF signaling by disruption of DNA binding.

“Phosphorylation inhibits LEF-1/TCF signaling by disruption of DNA binding (13)”

• cites [13] sourceNot checked — no full text of the source

Xenopus NLK is expressed ubiquitously as both a maternal and a zygotic transcript in Xenopus embryos.

“Since Xenopus NLK is expressed ubiquitously as both a maternal and a zygotic transcript in Xenopus embryos (11)”

• cites [11] sourceNot checked — no full text of the source

Phosphorylation inhibits LEF-1/TCF signaling by inducing redistribution of POP-1 from the nucleus to the cytoplasm.

“and by inducing redistribution of POP-1 from the nucleus to the cytoplasm (22).”

• cites [22] sourceNot checked — no full text of the source

β-catenin signaling through LEF-1/TCF is inhibited by several mechanisms at the level of LEF-1/TCF and β-catenin in the nucleus.

“Thus, it is likely that β-catenin signaling through LEF-1/TCF is inhibited by several mechanisms at the level of LEF-1/TCF and β-catenin in the nucleus.”

In both C. elegans and mammalian cells, the Nemo-like kinases LIT-1 and NLK, respectively, phosphorylate LEF-1/TCF.

“Furthermore, in both C. elegans and mammalian cells, the Nemo-like kinases LIT-1 and NLK, respectively, phosphorylate LEF-1/TCF.”

• cites [13] sourceNot checked — no full text of the source
• cites [22] sourceNot checked — no full text of the source

NLK phosphorylation inhibits β-catenin-mediated transcription through LEF-1/TCF-binding sites in Wnt pathway-responsive promoters.

“and thereby inhibits β-catenin-mediated transcription through LEF-1/TCF-binding sites in Wnt pathway-responsive promoters.”

• cites [13] sourceNot checked — no full text of the source

In vivo, coinjection of NLK RNA into early Xenopus embryos results in the inhibition of double-axis formation by β-catenin.

“In vivo, coinjection of NLK RNA into early Xenopus embryos results in the inhibition of double-axis formation by β-catenin.”

• cites [13] sourceNot checked — no full text of the source