TAB2 Scaffolds TAK1 and NLK in Repressing Canonical Wnt Signaling

Meng Li,He Wang,Tao Huang,Jiyong Wang,Yu Ding,Zhenfei Li,Jinkuo Zhang,Lin Li

doi:10.1074/jbc.m109.083246

Abstract

The TAK1-NLK cascade is a mitogen-activated protein kinase-related pathway that plays an inhibitory role in canonical Wnt/beta-catenin signaling through regulating the LEF1/TCF family transcriptional factors. TAB2 (TAK1-binding protein 2) is a putative TAK1 interacting protein that is involved in the regulation of TAK1. Here, we found that TAB2 could directly interact with NLK and function as a scaffold protein to facilitate the interaction between TAK1 and NLK. Knocking down TAB2 using small interfering RNA abolished the interaction of TAK1 with NLK in mammalian cells. The intermediate region (residues 292-417) of TAB2 was mapped for its binding to NLK. TAB2-DeltaM, a TAB2 mutant lacking this region, showed a lower affinity for NLK and became defective in its scaffolding function. In addition, TAB2, but not TAB2-DeltaM, mediated TAK1-dependent activation of NLK and LEF1 polyubiquitylation, resulting in the inhibition of canonical Wnt signaling. Moreover, Wnt3a stimulation led to an increase in the interaction of TAB2 with NLK and the formation of a TAK1.TAB2.NLK complex, suggesting that this TAK1-TAB2-NLK pathway may constitute a negative feedback mechanism for canonical Wnt signaling.

Highlights

TAK1 is the upstream activator of the kinase activity of NLK, which in turn phosphorylates and regulates several transcriptional factors [4, 6, 13], including lymphoid enhancer factor 1/T-cell factor (LEF1/TCF) family proteins [2, 14]
In this work we report that TAB2 functions as a scaffold protein for TAK1 and NLK to bridge their interaction in repressing canonical Wnt signaling
NLK Is Identified as a Novel TAB2-binding Protein—Previous studies have shown that TAB2 interacted with TAK1 in a number of TAK1-mediated signaling events [28, 29]

Summary

EXPERIMENTAL PROCEDURES

SiRNAs, and Antibodies—Full-length mouse NLK was identified from the pPC86-based mouse embryonic E10.5 cDNA library as a positive clone, which showed the interaction. TAB2, TAK1, and NLK antibodies were purchased from Santa Cruz Biotechnology, Inc. Anti-HA, Myc, and EE (Covance), antiFLAG (Sigma), anti-␤-catenin (BD Biosciences), and c-Jun N-terminal kinase (JNK) and phospho-JNK (Cell Signaling Technology) were used in this work. Plasmids were transfected using Lipofectamine/Plus reagent (Invitrogen) according to the manufacturer’s instructions. Cells were collected by spinning at 3000 rpm for 10 min and resuspended in buffer A (10 mM Hepes-KOH, pH 7.9, 10 mM KCl). Reverse Transcription-PCR and Quantitative Real-time PCR— Total RNAs were extracted from siRNA-transfected HEK293T cells with TRIzol, and reverse transcription of purified RNA was performed using oligo(dT) priming and superscript III reverse transcription according to the manufacturer’s instructions (Invitrogen). NLK Kinase Assay—HEK293T cells seeded in 35-mm dishes were transfected with indicated plasmids. For NLK kinase assays with NLK-293 cells, stably transfected NLK was pulled down with FLAG affinity gel (Sigma). Transfected cells were subjected to different assays 48 h after transfection

RESULTS AND DISCUSSION

In our work we also studied the

Lin Li