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Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop.

K Ohashi1, K Nagata, M Maekawa

  • 1Biological Institute, Graduate School of Science, Tohoku University, Sendai 980-8578, Japan.

The Journal of Biological Chemistry
|February 1, 2000
PubMed
Summary
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ROCK activates LIM-kinase 1 (LIMK1) by phosphorylating Thr-508, enhancing its ability to regulate actin cytoskeleton reorganization. This finding reveals a common activation pathway for LIMK1 involving Rho and Rac signaling.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • LIM-kinase 1 (LIMK1) regulates actin cytoskeleton dynamics by phosphorylating cofilin.
  • LIMK1 activation is known to involve the small GTPase Rho and its downstream kinase ROCK.

Purpose of the Study:

  • To identify the specific site of LIMK1 phosphorylation by ROCK.
  • To elucidate the mechanism by which ROCK activates LIMK1.
  • To understand the role of LIMK1 activation in Rho and Rac signaling pathways.

Main Methods:

  • In vitro kinase assays using wild-type and mutant LIMK1.
  • Site-directed mutagenesis of LIMK1 at Thr-508.
  • Co-expression of LIMK1 and ROCK in cultured cells.
  • Analysis of cofilin-phosphorylating activity.

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Main Results:

  • ROCK directly phosphorylates LIMK1 at Thr-508 in vitro.
  • Phosphorylation at Thr-508 by ROCK significantly increases LIMK1's cofilin-phosphorylating activity.
  • A LIMK1 mutant (T508A) is not phosphorylated or activated by ROCK.
  • ROCK activates wild-type LIMK1 in cultured cells, but not the T508A mutant.
  • Mutating Thr-508 to glutamate constitutively activates LIMK1 but prevents further ROCK-mediated activation.

Conclusions:

  • ROCK activates LIMK1 through phosphorylation at Thr-508, both in vitro and in vivo.
  • LIMK1 activation by phosphorylation at Thr-508 is a shared mechanism for Rho and Rac signaling in actin cytoskeleton reorganization.