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Phosphorylation by NLK inhibits YAP-14-3-3-interactions and induces its nuclear localization

Sungho Moon1, Wantae Kim1, Soyoung Kim1

  • 1Department of Life Science, University of Seoul, Seoul, Korea.

EMBO Reports
|December 17, 2016
PubMed

Insights

Nemo-like kinase (NLK) regulates organ size by controlling the nuclear localization of Yes-associated protein (YAP). NLK phosphorylation of YAP enhances its activity, suggesting a novel mechanism in Hippo signaling.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Developmental Biology

Background:

  • Hippo signaling is crucial for organ size regulation, controlling cell proliferation and apoptosis.
  • Yes-associated protein (YAP) is a key downstream effector of Hippo signaling, with LATS-mediated phosphorylation at Ser127 inhibiting its activity.
  • Understanding YAP regulation is vital for comprehending developmental processes and diseases.

Purpose of the Study:

  • To investigate the role of Nemo-like kinase (NLK) in regulating YAP activity and Hippo signaling.
  • To elucidate the specific phosphorylation sites on YAP targeted by NLK and their functional consequences.
  • To explore the in vivo relevance of NLK-YAP interactions in both mammalian and Drosophila models.

Main Methods:

  • In vitro and in vivo phosphorylation assays to determine NLK's effect on YAP.
  • Western blotting and immunoprecipitation to assess YAP phosphorylation and protein interactions.
  • Reporter gene assays to measure YAP transcriptional activity.
  • Gene knockdown and overexpression studies in mammalian cells and Drosophila wing imaginal discs.

Main Results:

  • NLK phosphorylates YAP at Ser128, promoting its nuclear localization and blocking 14-3-3 interaction.
  • NLK depletion increases YAP phosphorylation at Ser127, reducing YAP activity, suggesting mutually exclusive phosphorylation events.
  • NLK levels and nuclear localization decrease with increasing cell density, correlating with reduced YAP phosphorylation at Ser128.
  • Drosophila Nemo knockdown reduces expression of YAP/Yorkie target genes, while overexpression has reciprocal effects.

Conclusions:

  • NLK acts as an endogenous regulator of Hippo signaling by modulating YAP/Yorkie nuclear localization and activity.
  • NLK-mediated phosphorylation of YAP at Ser128 is a novel mechanism controlling YAP function.
  • The findings reveal a conserved role for NLK/Nemo in regulating Hippo pathway output across species.
Keywords:
NLKYAP14‐3‐3Hippo signalingcell density

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