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Identification of Kinase-substrate Pairs Using High Throughput Screening
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A Screen for PKN3 Substrates Reveals an Activating Phosphorylation of ARHGAP18

Michal Dibus1,2, Jan Brábek1,2, Daniel Rösel1,2

  • 1Department of Cell Biology, Charles University, Viničná 7, 12800 Prague, Czech Republic.

International Journal of Molecular Sciences
|October 23, 2020
PubMed

Insights

Researchers identified new protein kinase N3 (PKN3) substrates, revealing a feedback loop where PKN3 activates ARHGAP18 to regulate RhoA signaling, crucial for understanding cancer progression.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Cancer Research

Background:

  • Protein kinase N3 (PKN3) is a serine/threonine kinase involved in tumor progression.
  • PKN3's substrates and effector proteins are not well understood, limiting therapeutic target identification.

Purpose of the Study:

  • To identify novel substrates of PKN3 using phosphoproteomic screening.
  • To investigate the functional relationship between PKN3 and its newly identified substrate, ARHGAP18.

Main Methods:

  • Utilized analog-sensitive PKN3 for phosphoproteomic screening.
  • Performed in vitro kinase assays to validate PKN3-ARHGAP18 interaction and phosphorylation.
  • Assessed the impact of PKN3-mediated phosphorylation on ARHGAP18's Rho GTPase-activating protein (GAP) activity.

Main Results:

  • Identified ARHGAP18 as a novel substrate of PKN3.
  • Confirmed direct phosphorylation of ARHGAP18 by PKN3 in vitro, with interaction mediated by ARHGAP18's N-terminal region.
  • Demonstrated that PKN3 phosphorylation enhances ARHGAP18's GAP activity, leading to negative regulation of active RhoA.

Conclusions:

  • Discovered a new set of potential PKN3 substrates.
  • Elucidated a novel negative feedback mechanism in Rho signaling involving PKN3-induced activation of ARHGAP18.
  • This finding offers insights into cancer progression pathways regulated by PKN3 and RhoA.
Keywords:
ARHGAP18PKN3Rho-GTPphosphoproteomic screenphosphorylation

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