Phosphorylation of AFAP-110 affects podosome lifespan in A7r5 cells.
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.
Dorfleutner
Andrea
A
The Mary Babb Randolph Cancer Center and Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506-9300, USA.
Cho
Youngjin
Y
Vincent
Deanne
D
Cunnick
Jess
J
Lin
Hong
H
Weed
Scott A
SA
Stehlik
Christian
C
Flynn
Daniel C
DC
D000595:Amino Acid Sequence; D000818:Animals; D045163:Antibodies, Phospho-Specific; D019556:COS Cells; D002452:Cell Count; D022082:Cellular Structures / Q000378:metabolism*; D002522:Chlorocebus aethiops; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins; D009154:Mutation / Q000235:genetics; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D051571:Protein Kinase C-alpha / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
C084510:AFAP 110; D045163:Antibodies, Phospho-Specific; D008840:Microfilament Proteins; D050505:Mutant Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D010768:Phosphoserine; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate