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Evolution of CASK into a Mg2+-sensitive kinase.

Konark Mukherjee1, Manu Sharma, Reinhard Jahn

  • 1Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 1050 Arastradero Road, Palo Alto, CA 94304, USA. konark@brandeis.edu

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|April 29, 2010
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Summary

Researchers converted the magnesium-inhibited CASK kinase into a magnesium-stimulated enzyme by altering its ATP-binding pocket. This study reveals evolutionary insights into CASK regulation and its link to nervous system development.

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Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Evolutionary Biology

Background:

  • Most protein kinases require magnesium ions (Mg2+) for activity, utilizing them in phosphoryl transfer from ATP.
  • CASK (calcium/calmodulin-activated serine-threonine kinase) is an exception, with its kinase domain inhibited by Mg2+.

Purpose of the Study:

  • To investigate the structural and functional basis of Mg2+ inhibition in CASK.
  • To engineer a Mg2+-stimulated CASK variant and explore its catalytic mechanism.
  • To understand the evolutionary history of CASK's Mg2+ sensitivity.

Main Methods:

  • Site-directed mutagenesis to convert wild-type CASK (CASK(WT)) to a Mg2+-stimulated form (CASK(4M)).
  • X-ray crystallography to determine the structures of CASK(4M) with and without nucleotide and Mn2+.
  • Kinetic analyses to characterize the catalytic mechanism of CASK(4M).
  • Phylogenetic analysis to trace the evolutionary changes in CASK.

Main Results:

  • Four residue substitutions in the ATP-binding pocket converted Mg2+-inhibited CASK(WT) into Mg2+-stimulated CASK(4M).
  • Crystal structures revealed Mg2+ stabilizes the transition state, enhances ADP leaving group properties, and optimizes ATP gamma-phosphate positioning in CASK(4M).
  • Phylogenetic analysis indicates CASK evolved Mg2+ sensitivity (inhibition) early in animal history, coinciding with nervous system development.

Conclusions:

  • Mg2+ can accelerate CASK catalysis through specific transition state stabilization, contrary to its inhibitory effect on wild-type CASK.
  • The evolution of Mg2+ sensitivity in CASK provided a regulatory mechanism responsive to divalent cations, paralleling nervous system evolution.
  • Understanding CASK's unique Mg2+ interaction offers insights into kinase regulation and evolutionary adaptation.