Phosphorylation of AFAP-110 affects podosome lifespan in A7r5 cells

Andrea Dorfleutner, Young Jin Cho, Deanne Vincent, Jess Cunnick, Hong Lin, Scott A. Weed, Christian Stehlik, Daniel C. Flynn*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

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 PKCα, AFAP-110 is a substrate of PKCα and PKCα-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 PKCα 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-110S277A) did not alter the ability of AFAP-110 to localize to podosomes, overexpression of AFAP-110S277A in treated and untreated A7r5 cells resulted in an increased number of cells that display podosomes. Video microscopy demonstrated that AFAP-110S277A 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.

Original languageEnglish (US)
Pages (from-to)2394-2405
Number of pages12
JournalJournal of cell science
Volume121
Issue number14
DOIs
StatePublished - Jul 15 2008

Keywords

  • AFAP-110
  • AFAP1
  • Actin filaments
  • Invadopodia
  • PKC
  • Podosomes

ASJC Scopus subject areas

  • Cell Biology

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