FAP52 regulates actin organization via binding to filamin.
FAP52, a focal adhesion-associated phosphoprotein, is a member of a FAP52/PACSIN/syndapin family of proteins. They share a multidomain structure and are implicated in actin-based and endocytotic functions. We show, by using both native and recombinant proteins, that FAP52 selectively binds to the actin cross-linking protein filamin (ABP-280). This was based on an affinity purification followed by a sequence determination by mass spectrometry, co-immunoprecipitation, overlay binding, and surface plasmon resonance analysis. Binding studies with deletion mutants showed that the sites of the interaction map to the highly alpha-helical N-terminal part of FAP52 and to the C-terminal region of filamin, which also contains binding sites to some transmembrane signaling proteins. In immunofluorescence and immunoelectron microscopy of cultured fibroblasts, a different overall subcellular distribution was seen for filamin and FAP52 except for a stress fiber-focal adhesion junction where they showed a notable overlap. Overexpression of the full-length and mutant forms of FAP52 led to an extensive reorganization of actin and filamin in cultured fibroblasts. Thus, the results show that FAP52 interacts with filamin, and we propose that this interaction is important in linking and coordinating the events between focal adhesions and the actin cytoskeleton.
Nikki
Marko
M
Department of Pathology, University of Oulu, FIN-90014 Oulu, Finland.
Meriläinen
Jari
J
Lehto
Veli-Pekka
VP
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D002846:Chromatography, Affinity; D003285:Contractile Proteins / Q000378:metabolism*; D064448:Filamins; D022001:Focal Adhesions; D008840:Microfilament Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D016296:Mutagenesis; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D020349:Surface Plasmon Resonance
D000199:Actins; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; C108082:focal adhesion protein, 52-kDa