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Phosphorylation of RhoB by CK1 impedes actin stress fiber organization and epidermal growth factor receptor

Vanessa Tillement1, Isabelle Lajoie-Mazenc, Anne Casanova

  • 1INSERM, U563, CPTP, Département Innovation Thérapeutique et Oncologie Moléculaire, Toulouse F-31052, France.

Experimental Cell Research
|July 2, 2008
PubMed

Insights

This study reveals that Casein Kinase 1 (CK1) phosphorylates RhoB, a small GTPase, at Serine 185. Inhibiting CK1 activates RhoB, impacting actin fibers and EGF receptor levels.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • RhoB, a small GTPase, plays a role in cytoskeletal organization, EGF receptor trafficking, and cell transformation.
  • RhoB is an immediate-early gene regulated at multiple biosynthetic levels.

Purpose of the Study:

  • To investigate the post-translational modification of RhoB.
  • To identify the kinase responsible for RhoB phosphorylation and its functional consequences.

Main Methods:

  • In vitro kinase assays using purified proteins.
  • Cellular phosphorylation studies in HeLa cells using CK1 inhibitors (IC261, D4476).
  • Mass spectrometry for phosphosite identification and site-directed mutagenesis (Ser185 to Alanine).

Main Results:

  • Casein Kinase 1 (CK1) specifically phosphorylates RhoB, but not RhoA or RhoC, in vitro and in HeLa cells.
  • Mass spectrometry identified Serine 185 in the C-terminal end of RhoB as the monophosphorylation site by CK1.
  • Mutation of Ser185 to Alanine significantly reduced RhoB phosphorylation in cells.
  • CK1 inhibition led to RhoB activation, promoting actin fiber formation and increasing EGF-R levels.

Conclusions:

  • This study provides the first evidence of RhoB phosphorylation.
  • CK1-mediated phosphorylation of RhoB at Serine 185 is a novel post-translational modification.
  • This phosphorylation acts as a critical regulatory mechanism controlling RhoB functions, including actin organization and EGF-R signaling.

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