Issues associated with assessing nuclear localization of N-terminally unphosphorylated β-catenin with monoclonal antibody 8E7

Maher, Meghan T.; Flozak, Annette S.; Hartsell, Alyssa M; Russell, Susan; Beri, Rohinee; Peled, Ofra N.; Gottardi, Cara J.

doi:10.1186/1745-6150-4-5

Cited by 20 publications

(15 citation statements)

References 19 publications

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“…It is possible some discrepancies arise from the antibody originally used to recognize dephospho-␤-catenin, which we (supplemental Fig. S8A) and others have found to be nonspecific (51).…”

Section: Discussionmentioning

confidence: 98%

Specific Armadillo Repeat Sequences Facilitate β-Catenin Nuclear Transport in Live Cells via Direct Binding to Nucleoporins Nup62, Nup153, and RanBP2/Nup358

Sharma

Jamieson

Johnson

et al. 2012

Journal of Biological Chemistry

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Background:The nuclear localization of ␤-catenin is directly linked to its cancer causing activity. Results: Armadillo repeats (10 -12) mediate nuclear transport of ␤-catenin through direct interaction with specific nuclear pore complex proteins. Conclusion: ␤-Catenin can function like a nuclear transport receptor in its ability to translocate independently through the nuclear pore complex. Significance: ␤-Catenin may transport specific binding partners between the nucleus and cytoplasm in response to Wnt signaling.␤-Catenin transduces the Wnt signal from the membrane to nucleus, and certain gene mutations trigger its nuclear accumulation leading to cell transformation and cancer. ␤-Catenin shuttles between the nucleus and cytoplasm independent of classical Ran/transport receptor pathways, and this movement was previously hypothesized to involve the central Armadillo (Arm) domain. Fluorescence recovery after photobleaching (FRAP) assays were used to delineate functional transport regions of the Arm domain in living cells. The strongest nuclear import/export activity was mapped to Arm repeats R10 -12 using both in vivo FRAP and in vitro export assays. By comparison, Arm repeats R3-8 of ␤-catenin were highly active for nuclear import but displayed a comparatively weak export activity. We show for the first time using purified components that specific Arm sequences of ␤-catenin interact directly in vitro with the FG repeats of the nuclear pore complex (NPC) components Nup62, Nup98, and Nup153, indicating an independent ability of ␤-catenin to traverse the NPC. Moreover, a proteomics screen identified RanBP2/Nup358 as a binding partner of Arm R10 -12, and ␤-catenin was confirmed to interact with endogenous and ectopic forms of Nup358. We further demonstrate that knock-down of endogenous Nup358 and Nup62 impeded the rate of nuclear import/export of ␤-catenin to a greater extent than that of importin-␤. The Arm R10 -12 sequence facilitated transport even when ␤-catenin was bound to the Arm-binding partner LEF-1, and its activity was stimulated by phosphorylation at Tyr-654. These findings provide functional evidence that the Arm domain contributes to regulated ␤-catenin transport through direct interaction with the NPC.The canonical Wnt signaling pathway regulates normal tissue development and maintenance through its effector molecule, ␤-catenin. In the absence of Wnt signaling ␤-catenin accumulates at adherens junctions. ␤-Catenin levels are regulated through degradation by a destruction complex, comprising of adenomatous polyposis coli (APC), 4 axin, casein kinase I, and glycogen synthase kinase-3␤, which phosphorylates and subsequently ubiquitinates ␤-catenin at the N terminus, leading to its degradation by the proteosome (1, 2). On receiving Wnt signals or through misregulation of the degradation pathway, ␤-catenin is stabilized and accumulates in the nucleus where it binds to lymphoid enhancer factor-1 (LEF/ TCF) transcription factors and activates transcription of tumor promoting genes involved in cell migration...

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Section: Discussionmentioning

confidence: 98%

Specific Armadillo Repeat Sequences Facilitate β-Catenin Nuclear Transport in Live Cells via Direct Binding to Nucleoporins Nup62, Nup153, and RanBP2/Nup358

Sharma

Jamieson

Johnson

et al. 2012

Journal of Biological Chemistry

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“…Previous studies showed that LiCl treatment of cells stimulated the nuclear transactivation potential of β‐catenin by increasing the pool of N‐terminal dephosphorylated β‐catenin (10,13). However, a recent study has disputed the specificity of the antibody used to detect nuclear dephosphorylated β‐catenin (14). To test whether the dephosphorylation of β‐catenin alters its nuclear dynamics, we employed a β‐catenin cDNA expressing the S45A mutation, which abolishes the CK1 priming phosphorylation and subsequent GSK‐3β‐dependent phosphorylation of β‐catenin, and thus should mimic the effect of LiCl and increase nuclear retention of β‐catenin.…”

Section: Resultsmentioning

confidence: 99%

Regulation of β‐Catenin Nuclear Dynamics by GSK‐3β Involves a LEF‐1 Positive Feedback Loop

Jamieson

Sharma

Henderson

2011

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Nuclear localization of β-catenin is integral to its role in Wnt signaling and cancer. Cellular stimulation by Wnt or lithium chloride (LiCl) inactivates glycogen synthase kinase-3β (GSK-3β), causing nuclear accumulation of β-catenin and transactivation of genes that transform cells. β-catenin is a shuttling protein; however, the mechanism by which GSK-3β regulates β-catenin nuclear dynamics is poorly understood. Here, fluorescence recovery after photobleaching assays were used to measure the β-catenin-green fluorescent protein dynamics in NIH 3T3 cells before and after GSK-3β inhibition. We show for the first time that LiCl and Wnt3a cause a specific increase in β-catenin nuclear retention in live cells and in fixed cells after detergent extraction. Moreover, LiCl reduced the rate of nuclear export but did not affect import, hence biasing β-catenin transport toward the nucleus. Interestingly, the S45A mutation, which blocks β-catenin phosphorylation by GSK-3β, did not alter nuclear retention or transport, implying that GSK-3β acts through an independent regulator. We compared five nuclear binding partners and identified LEF-1 as the key mediator of Wnt3a and LiCl-induced nuclear retention of β-catenin. The canonical Wnt/β-catenin signaling pathway controls the transcription of genes that function during normal and malignant development (1,2). Signaling through β-catenin is regulated by modulation of its degradation and nuclear translocation. In the absence of Wnt signaling, cellular β-catenin is tightly maintained at low levels via rapid degradation by the ubiquitin-proteasome system. This normal regulation involves phosphorylation of the β-catenin N-terminus by casein kinase I (CK1) at S45, priming the sequential phosphorylation of T41, S37 and S33 by glycogen synthase kinase-3β (GSK-3β) in a multiprotein complex comprising adenomatous polyposis coli (APC) protein and axin (1,2). The phosphorylation of β-catenin tags it for ubiquitination and proteosome-mediated degradation. In the presence of Wnt ligand, however, a complex between Frizzled receptor and LRP5/6 is formed that disrupts axin-GSK3β association required for phosphorylation of β-catenin, resulting in its stabilization and translocation into the nucleus (3). Similarly, mutations in the APC, axin or β-catenin genes (4,5), likewise lead to nuclear accumulation of stable β-catenin. In the nucleus, β-catenin binds and activates lymphoid enhancer-binding factor-1/T-cell factor (LEF-1/TCF) and other transcription factors (6) leading to stimulated transcription of Wnt target genes frequently misregulated in human tumors including cyclin D1, c-myc and metalloproteases (1,7,8).Immunohistochemical studies on human tumors reveal increased nuclear β-catenin at the invasive front of differentiated mesenchyme-like tumors, whereas in the central areas of primary tumors β-catenin is located at membrane junctions and cytoplasm (8). This localization pattern can be a useful diagnostic tool, in that a study on colorectal cancers described a progressive increase in nucl...

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“…Because AXIN2 expression can be significantly (42). The nuclear staining can be unspecific as described (70). D, Wnt/␤-catenin signaling activation in mouse AT2 cells isolated from AXIN2 ϩ/LacZ reporter mice, but not AXIN2 ϩ/ϩ mice.…”

Section: Endogenous Activation Of ␤-Catenin/tcf Signaling Activity Inmentioning

confidence: 99%

β-Catenin/T-cell Factor Signaling Is Activated during Lung Injury and Promotes the Survival and Migration of Alveolar Epithelial Cells

Flozak

Lam

Russell

et al. 2010

Journal of Biological Chemistry

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The Wnt/␤-catenin signaling cascade activates genes that allow cells to adopt particular identities throughout development. In adult self-renewing tissues like intestine and blood, activation of the Wnt pathway maintains a progenitor phenotype, whereas forced inhibition of this pathway promotes differentiation. In the lung alveolus, type 2 epithelial cells (AT2) have been described as progenitors for the type 1 cell (AT1), but whether AT2 progenitors use the same signaling mechanisms to control differentiation as rapidly renewing tissues is not known. We show that adult AT2 cells do not exhibit constitutive ␤-catenin signaling in vivo, using the AXIN2

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Issues associated with assessing nuclear localization of N-terminally unphosphorylated β-catenin with monoclonal antibody 8E7

Cited by 20 publications

References 19 publications

Specific Armadillo Repeat Sequences Facilitate β-Catenin Nuclear Transport in Live Cells via Direct Binding to Nucleoporins Nup62, Nup153, and RanBP2/Nup358

Specific Armadillo Repeat Sequences Facilitate β-Catenin Nuclear Transport in Live Cells via Direct Binding to Nucleoporins Nup62, Nup153, and RanBP2/Nup358

Regulation of β‐Catenin Nuclear Dynamics by GSK‐3β Involves a LEF‐1 Positive Feedback Loop

β-Catenin/T-cell Factor Signaling Is Activated during Lung Injury and Promotes the Survival and Migration of Alveolar Epithelial Cells

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