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The CDC42 effector protein MRCKβ autophosphorylates on Threonine 1108

Mathieu Unbekandt1, Sergio Lilla1, Sara Zanivan1

  • 1Cancer Research UK Beatson Institute , Glasgow, UK.

Small Gtpases
|January 23, 2019
PubMed

Insights

Researchers identified a new autophosphorylation site, Threonine 1108, on Myotonic dystrophy related CDC42-binding kinase β (MRCKβ). This site does not appear to affect MRCKβ function but may serve as a biomarker for kinase activity.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • CDC42 small GTPase regulates cytoskeleton organization via effector proteins like Myotonic dystrophy related CDC42-binding kinases (MRCK) α and β.
  • Previous work identified Serine 1003 on MRCKα as an autophosphorylation site, useful as a kinase activity biomarker.

Purpose of the Study:

  • To identify novel phosphorylation sites on MRCKβ, particularly those resulting from autophosphorylation.
  • To investigate the functional and regulatory roles of a newly identified phosphorylation site, Threonine 1108 (Thr1108), on MRCKβ.

Main Methods:

  • Established a kinase-dead MRCKβ mutant (K105M) using site-directed mutagenesis.
  • Utilized mass spectrometry to compare phosphorylation events between wild-type MRCKβ and the kinase-dead mutant.
  • Created Thr1108 alanine (T1108A) and glutamate (T1108E) mutants to assess phosphorylation effects.

Main Results:

  • Identified ten phosphorylation sites on wild-type MRCKβ, including Thr1108, which was absent in the kinase-dead mutant.
  • Mutation of Thr1108 did not alter MRCKβ's in vitro myosin light chain phosphorylation activity.
  • Subcellular localization of GFP-tagged MRCKβ in breast cancer cells remained unchanged with Thr1108 mutations.

Conclusions:

  • Thr1108 is a novel autophosphorylation site on MRCKβ.
  • Phosphorylation at Thr1108 does not significantly impact MRCKβ's kinase activity or cellular localization.
  • Thr1108 warrants further investigation as a potential surrogate biomarker for MRCKβ activity and inhibitor efficacy.
Keywords:
CDC42MRCKkinasephosphorylation

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