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First published online 24 June 2008
doi: 10.1242/jcs.026187

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Phosphorylation of AFAP-110 affects podosome lifespan in A7r5 cells

Andrea Dorfleutner^1,2,*, YoungJin Cho^1,2, Deanne Vincent^1,2, Jess Cunnick^1,3, Hong Lin^1,2, Scott A. Weed^1,4, Christian Stehlik^1,2,* and Daniel C. Flynn^1,2,

¹ The Mary Babb Randolph Cancer Center and Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506-9300, USA
² The Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV, USA
³ The Department of Pathology, West Virginia University, Morgantown, WV, USA
⁴ The Department of Neurobiology and Anatomy, West Virginia University, Morgantown, WV, USA

Author for correspondence (e-mail: dflynn{at}hsc.wvu.edu)

Accepted 24 April 2008

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 PKC, AFAP-110 is a substrate of PKC and PKC–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 PKC 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.

Key words: AFAP1, AFAP-110, Podosomes, PKC, Invadopodia, Actin filaments

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