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Erk1/2 MAPK and caldesmon differentially regulate podosome dynamics in A7r5 vascular smooth muscle cells

Zhizhan Gu1, Jolanta Kordowska, Geoffrey L Williams

  • 1Department of Molecular Pharmacology, Physiology and Biotechnology, Box G-B3, Brown University, Providence, RI 02912, USA.

Experimental Cell Research
|January 24, 2007
PubMed

Insights

Protein kinase C (PKC)-mediated podosome dynamics in A7r5 cells are regulated by the MEK/Erk/caldesmon pathway. Caldesmon phosphorylation influences podosome size and lifetime, impacting vascular smooth muscle cell function.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Podosomes are dynamic actin-rich structures crucial for cell migration and invasion.
  • Protein kinase C (PKC) signaling plays a role in regulating podosome formation and dynamics.
  • The MEK/Erk/caldesmon phosphorylation cascade is implicated in various cellular processes.

Purpose of the Study:

  • To investigate the role of the MEK/Erk/caldesmon phosphorylation cascade in regulating PKC-mediated podosome dynamics in A7r5 cells.
  • To elucidate the specific contributions of Erk and caldesmon phosphorylation to podosome structure and function.

Main Methods:

  • Stimulation of A7r5 cells with phorbol dibutyrate (PDBu) to induce podosome formation.
  • Inhibition of MEK using U0126 to assess Erk translocation and phosphorylation.
  • Live-imaging microscopy to observe podosome dynamics and column formation.
  • Analysis of protein expression and localization (e.g., alpha-actinin, caldesmon, vinculin, metalloproteinase-2).
  • Overexpression of wild-type and phosphorylation-deficient caldesmon-GFP mutants.

Main Results:

  • Phorbol dibutyrate stimulation induced phosphorylation and translocation of MEK, Erk, and caldesmon to podosomes, with nuclear translocation of phospho-MEK and phospho-Erk.
  • MEK inhibition prevented Erk nuclear translocation, suggesting podosomes are sites of Erk phosphorylation.
  • Interconnected actin-rich columns formed, containing alpha-actinin, caldesmon, vinculin, and metalloproteinase-2.
  • Caldesmon and vinculin integrated with F-actin at these columns, differing from their usual ring localization.
  • Live imaging revealed column growth from slowly disassembling podosomes.
  • Caldesmon and its phosphorylation modulated podosome size and lifetime in A7r5 cells.

Conclusions:

  • The MEK/Erk/caldesmon phosphorylation cascade is a key regulator of PKC-mediated podosome dynamics in A7r5 vascular smooth muscle cells.
  • Erk1/2 and caldesmon differentially modulate the formation and/or dynamics of podosomes.
  • Podosomes serve as platforms for Erk phosphorylation and contribute to the formation of larger actin-rich structures.

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