Related Experiment Video

Updated: Jun 4, 2026

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation
09:40

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Published on: June 27, 2017

Phosphorylation of TCF proteins by homeodomain-interacting protein kinase 2

Hiroki Hikasa1, Sergei Y Sokol

  • 1Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, New York 10029, USA.

The Journal of Biological Chemistry
|February 3, 2011
PubMed

Insights

Homeodomain-interacting protein kinase 2 (HIPK2) regulates Wnt signaling by phosphorylating TCF proteins in Xenopus embryos. This phosphorylation affects gene transcription by altering TCF protein binding to target promoters.

Area of Science:

  • Developmental Biology
  • Molecular Biology
  • Cell Signaling

Background:

  • Wnt pathways are crucial for embryonic development, controlling cell proliferation, morphogenesis, and fate.
  • The canonical Wnt pathway involves stabilizing beta-catenin, which then interacts with TCF proteins to regulate gene expression.
  • The precise roles of specific regulators like HIPK2 in modulating TCF activity within the Wnt pathway are still being elucidated.

Purpose of the Study:

  • To investigate the role of HIPK2 in regulating TCF proteins during Wnt signaling in Xenopus embryos.
  • To understand how HIPK2-mediated phosphorylation of TCF proteins influences their interaction with target gene promoters.
  • To elucidate the context-dependent functions of HIPK2 in Wnt-mediated transcriptional regulation.

Main Methods:

  • Utilized Xenopus embryos for in vivo studies.
  • Stimulated Wnt signaling using Wnt8 and overexpressed HIPK2.
  • Analyzed phosphorylation of TCF family members (LEF1, TCF4, TCF3) using Western blotting or similar techniques.
  • Assessed TCF protein binding to target promoters in vivo.
  • Investigated the exchange of TCF3 with TCF1 at target promoters.

Main Results:

  • TCF family members LEF1, TCF4, and TCF3 were phosphorylated in response to Wnt8 stimulation and HIPK2 overexpression in embryonic ectoderm.
  • TCF3 phosphorylation was regulated by canonical Wnt ligands, LRP6, and dominant-negative Axin/GSK3 mutants, linking it to beta-catenin stabilization.
  • HIPK2-dependent phosphorylation led to the dissociation of LEF1, TCF4, and TCF3 from target promoters.
  • HIPK2 exhibited context-dependent transcriptional regulation: up-regulating genes via TCF3 (repressor) and down-regulating via LEF1 (activator).
  • Phosphorylated TCF3 was replaced by TCF1 at target promoters upon HIPK2 action.

Conclusions:

  • HIPK2 plays a critical role in Wnt signaling through the phosphorylation of TCF proteins in vertebrate embryos.
  • TCF phosphorylation by HIPK2 modulates TCF binding to DNA, impacting Wnt target gene transcription.
  • TCF switching, specifically the replacement of TCF3 with TCF1, is a key mechanism for Wnt target gene activation mediated by Wnt/HIPK2 signaling.

More Related Videos

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors08:45

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors

Published on: July 17, 2020

Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis07:26

Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Published on: April 1, 2022

Related Concept Videos

TGF - β Signaling Pathway01:16

TGF - β Signaling Pathway

The TGF-β signaling pathway regulates cell growth, differentiation, adhesion, motility, and development. TGF-β ligands that induce TGF-β signaling are synthesized in their latent form. Several proteases or cell surface receptors such as integrins act upon the latent form, releasing the active ligand. There are three types of mammalian TGF-βs: (TGF-β1, TGF-β2, and TGF-β3) that bind as homodimers or heterodimers to TGF-β receptors. The TGF-β receptors are of three kinds RI, RII, and RIII. The RI...
The JAK-STAT Signaling Pathway01:20

The JAK-STAT Signaling Pathway

Several cytokine receptors have tightly bound Janus kinase or JAK proteins attached at their cytosolic tail. Small signaling molecules such as cytokines, growth hormones, or prolactins bind to the cytokine receptors and initiate their dimerization. The dimerization brings the cytosolic JAKs together that trans-phosphorylate and activates each other. The activated JAKs now phosphorylate cytosolic tails of the cytokine receptors, which serve as binding sites for adaptor proteins such as  SH2...
PI3K/mTOR/AKT Signaling Pathway01:22

PI3K/mTOR/AKT Signaling Pathway

The mammalian target of rapamycin  (mTOR) is a serine/threonine kinase that regulates growth, proliferation, and cell survival in response to hormones, growth factors, or nutrient availability. This kinase exists in two structurally and functionally distinct forms: mTOR complex 1  (mTORC1) and mTOR complex 2  (mTORC2). The first form (mTORC1) is composed of a rapamycin-sensitive Raptor and proline-rich Akt substrate, PRAS40. In contrast,  mTORC2 consists of a rapamycin-insensitive companion...
Calmodulin-dependent Signaling01:16

Calmodulin-dependent Signaling

Calmodulin (CaM) is a calcium-binding protein in eukaryotes that controls various calcium-regulated cellular processes. It has four calcium-binding sites that bind calcium to form the calcium-calmodulin ( Ca2+-CaM) complex. GPCR stimulation increases the calcium levels in the cells that bind to CaM and induces a conformational change.
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...
MAPK Signaling Cascades01:07

MAPK Signaling Cascades

Mitogen-activated protein kinase, or MAPK pathway, activates three sequential kinases to regulate cellular responses such as proliferation, differentiation, survival, and apoptosis. The canonical MAPK pathway starts with a mitogen or growth factor binding to an RTK. The activated RTKs stimulate Ras, which recruits Raf or MAP3 Kinase (MAPKKK), the first kinase of the MAPK signaling cascade. Raf further phosphorylates and activates MEK or MAP2 Kinases (MAPKK), which in turn phosphorylates MAP...
Receptor Tyrosine Kinases01:26

Receptor Tyrosine Kinases

Receptor tyrosine kinases or RTKs are membrane-bound receptors that phosphorylate specific tyrosine on protein substrates. RTKs regulate cellular growth, differentiation, survival, and migration. They contain an extracellular ligand binding domain, a transmembrane domain, and a cytosolic tail with intrinsic kinase activity. Several extracellular signaling molecules activate RTKs in one or more ways and relay the signal downstream. Ligands such as platelet-derived growth factor (PDGF) or...