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Identification of Kinase-substrate Pairs Using High Throughput Screening
11:13

Identification of Kinase-substrate Pairs Using High Throughput Screening

Published on: August 29, 2015

Substrate recognition domains within extracellular signal-regulated kinase mediate binding and catalytic activation

A Nichols1, M Camps, C Gillieron

  • 1Serono Pharmaceutical Research Institute, Ares-Serono International SA, Plan-les-Ouates 1228, Geneva, Switzerland.

The Journal of Biological Chemistry
|May 17, 2000
PubMed

Insights

Mitogen-activated protein kinase phosphatase-3 (MKP-3) inactivates extracellular signal-regulated kinase (ERK) through specific binding. The COOH-terminal lobe of ERK, comprising subdomains V-XI, is crucial for this interaction and phosphatase activation.

Area of Science:

  • Molecular Biology
  • Enzymology
  • Signal Transduction

Background:

  • Mitogen-activated protein kinase phosphatase-3 (MKP-3) is a key enzyme regulating extracellular signal-regulated kinase (ERK) activity.
  • MKP-3 deactivates ERK through specific binding and subsequent dephosphorylation.

Purpose of the Study:

  • To identify the specific domains within ERK essential for binding and catalytic activation by MKP-3.
  • To elucidate the structural basis of the interaction between ERK and MKP-3.

Main Methods:

  • Utilized p38/ERK chimeric molecules to map functional domains.
  • Investigated the role of specific ERK subdomains (V-XI) in MKP-3 interaction.
  • Analyzed the effect of substrate-mimicking peptides and MKP-3 mutations on phosphatase activation.

Main Results:

  • ERK kinase subdomains V-XI were identified as necessary and sufficient for MKP-3 binding and activation.
  • These domains form the major COOH-terminal structural lobe of ERK.
  • Chimeric ERK molecules containing these domains showed enhanced sensitivity to MKP-3 inactivation.
  • Overlap was observed between ERK domains interacting with MKP-3 and those conferring substrate specificity.
  • Peptides from target substrates (Elk-1, p90rsk) and mutations in MKP-3's kinase interaction motif (KIM) inhibited ERK-dependent activation.

Conclusions:

  • The COOH-terminal structural lobe of ERK (subdomains V-XI) is critical for specific recognition and activation of MKP-3.
  • The interaction interface for MKP-3 involves residues within ERK's COOH-terminal lobe and potentially MKP-3's NH2-terminal KIM.
  • Understanding these interactions provides insights into MAP kinase regulation and substrate specificity.

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