Related Experiment Video

Updated: Jun 3, 2025

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes
09:40

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Published on: September 28, 2018

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Capture, mutual inhibition and release mechanism for aPKC-Par6 and its multisite polarity substrate Lgl

Christopher P Earl1, Mathias Cobbaut2,3, André Barros-Carvalho4,5

  • 1Signalling and Structural Biology Laboratory, Francis Crick Institute, London, UK.

Nature Structural & Molecular Biology
|January 6, 2025
PubMed

Insights

A novel structure reveals how atypical protein kinase C (aPKC) and partitioning-defective protein 6 (Par6) interact with lethal (2) giant larvae (Lgl) protein. This interaction regulates cell polarity by forming a stable complex that inhibits phosphorylation.

Area of Science:

  • Cell biology
  • Molecular biology
  • Structural biology

Background:

  • The atypical protein kinase C (aPKC)-partitioning-defective protein 6 (Par6) complex antagonizes lethal (2) giant larvae (Lgl) protein.
  • This interaction is crucial for maintaining cell polarity in various cell types.
  • The precise mechanism of this regulation, particularly the role of Par6, remains unclear.

Purpose of the Study:

  • To elucidate the structural basis of the aPKC-Par6-Lgl interaction.
  • To understand the mechanism of mutual regulation and spatial control of these proteins.
  • To investigate the role of Par6 in the kinase-substrate complex.

Main Methods:

  • X-ray crystallography to determine the structure of human aPKCι-Par6α bound to full-length Llgl1.
  • Site-directed mutagenesis to disrupt specific interaction sites.
  • Biochemical assays to assess phosphorylation and complex stability.

Main Results:

  • The structure reveals a stable complex mediated by an aPKCι docking site and a Par6PDZ contact.
  • A phospho-S663 Llgl1 intermediate bridges aPKC and Par6, inhibiting further phosphorylation and forming a substrate-trapped intermediate.
  • Disrupting Lgl-aPKC interaction impairs complex assembly, while disrupting Lgl-Par6PDZ interaction promotes dissociation and phosphorylation.

Conclusions:

  • A Par6PDZ-regulated substrate capture-and-release model explains the mutual regulation of aPKC-Par6 and Lgl.
  • Complex disassembly requires Cdc42 binding and the apical protein Crumbs.
  • This mechanism provides spatial control over cell polarity signaling.

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