Phosphorylation of AFAP-110 affects podosome lifespan in A7r5 cells

J Cell Sci. 2008 Jul 15;121(Pt 14):2394-405. doi: 10.1242/jcs.026187. Epub 2008 Jun 24.


AFAP-110 is an actin-binding and -crosslinking protein that is enriched in Src and phorbol ester (PE)-induced podosomes. In vascular smooth muscle cells endogenous AFAP-110 localized to actin stress fibers and, in response to treatment with phorbol-12,13-dibutyrate (PDBu), to actin-rich podosomes. Since PEs can activate PKCalpha, AFAP-110 is a substrate of PKCalpha and PKCalpha-AFAP-110 interactions direct podosome formation, we sought to identify a PE-induced phosphorylation site in AFAP-110 and determine whether phosphorylation is linked to the formation of podosomes. Mutational analysis revealed Ser277 of AFAP-110 to be phosphorylated in PE-treated cells. The use of a newly generated, phospho-specific antibody directed against phosphorylated Ser277 revealed that PKCalpha activation is associated with PE-induced AFAP-110 phosphorylation. In PDBu-treated A7r5 rat vascular smooth muscle cells, immunolabeling using the phospho-specific antibody showed that phospho-AFAP-110 is primarily associated with actin in podosomes. Although mutation of Ser at position 277 to Ala (AFAP-110(S277A)) did not alter the ability of AFAP-110 to localize to podosomes, overexpression of AFAP-110(S277A) in treated and untreated A7r5 cells resulted in an increased number of cells that display podosomes. Video microscopy demonstrated that AFAP-110(S277A) expression correlates with an increased number of long-lived podosomes. Therefore, we hypothesize that AFAP-110 phosphorylation and/or dephosphorylation is involved in the regulation of podosome stability and lifespan.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Phospho-Specific
  • COS Cells
  • Cell Count
  • Cellular Structures / metabolism*
  • Chlorocebus aethiops
  • Microfilament Proteins / chemistry
  • Microfilament Proteins / metabolism*
  • Molecular Sequence Data
  • Mutant Proteins
  • Mutation / genetics
  • Myocytes, Smooth Muscle / cytology
  • Myocytes, Smooth Muscle / enzymology
  • Myocytes, Smooth Muscle / metabolism*
  • Phosphoproteins / chemistry
  • Phosphoproteins / metabolism*
  • Phosphorylation / drug effects
  • Phosphoserine / metabolism
  • Protein Kinase C-alpha / metabolism
  • Protein Structure, Tertiary
  • Protein Transport / drug effects
  • Rats
  • Recombinant Fusion Proteins / metabolism
  • Tetradecanoylphorbol Acetate / pharmacology


  • AFAP 110
  • Antibodies, Phospho-Specific
  • Microfilament Proteins
  • Mutant Proteins
  • Phosphoproteins
  • Recombinant Fusion Proteins
  • Phosphoserine
  • Protein Kinase C-alpha
  • Tetradecanoylphorbol Acetate