Palladin is a novel binding partner for Ena/VASP family members.
Palladin is an actin-associated protein that contains proline-rich motifs within its amino-terminal sequence that are similar to motifs found in zyxin, vinculin, and the Listeria protein ActA. These motifs are known to be potential binding sites for the Vasodilator-Stimulated Phosphoprotein (VASP). Here, we demonstrate that palladin is an additional direct binding partner for VASP, by using co-immunoprecipitation and blot overlay techniques with both endogenous palladin and recombinant myc-tagged palladin. These results show that VASP binds to full-length palladin and also to the amino-terminal half of palladin, where the polyproline motifs are located. Using a synthetic peptide array, two discrete binding sites for VASP were identified within palladin's proline-rich amino-terminal domain. Using double-label immunofluorescence staining of fully-spread and actively-spreading fibroblasts, the extent of co-localization of palladin and VASP was explored. These proteins were found to strongly co-localize along stress fibers, and partially co-localize in focal adhesions, lamellipodia, and focal complexes. These results suggest that the recently described actin-associated protein palladin may play an important role in recruiting VASP to sites of actin filament growth, anchorage, and crosslinking.
Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, 27599-7545, USA.
Malika
M
Boukhelifa
Mana M
MM
Parast
James E
JE
Bear
Frank B
FB
Gertler
Carol A
CA
Otey
D000595:Amino Acid Sequence; D000818:Animals; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002642:Chick Embryo; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006023:Glycoproteins; D006801:Humans; D008667:Metalloproteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D016596:Vinculin / Q000235:genetics / Q000378:metabolism; D060589:Zyxin; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; C092744:ENA-VASP proteins; D006023:Glycoproteins; D008667:Metalloproteins; D008840:Microfilament Proteins; C412722:PALLD protein, human; D010750:Phosphoproteins; C092842:ZYX protein, human; C498285:Zyx protein, mouse; D060589:Zyxin; C485873:palladin protein, mouse; D000098662:Vasodilator-Stimulated Phosphoprotein; D016596:Vinculin