Identification, purification, and characterization of a zyxin-related protein that binds the focal adhesion and microfilament protein VASP (vasodilator-stimulated phosphoprotein).
VASP (vasodilator-stimulated phosphoprotein), an established substrate of cAMP- and cGMP-dependent protein kinases in vitro and in living cells, is associated with focal adhesions, microfilaments, and membrane regions of high dynamic activity. Here, the identification of an 83-kDa protein (p83) that specifically binds VASP in blot overlays of different cell homogenates is reported. With VASP overlays as a detection tool, p83 was purified from porcine platelets and used to generate monospecific polyclonal antibodies. VASP binding to purified p83 in solid-phase binding assays and the closely matching subcellular localization in double-label immunofluorescence analyses demonstrated that both proteins also directly interact as native proteins in vitro and possibly in living cells. The subcellular distribution, the biochemical properties, as well as microsequencing data revealed that porcine platelet p83 is related to chicken gizzard zyxin and most likely represents the mammalian equivalent of the chicken protein. The VASP-p83 interaction may contribute to the targeting of VASP to focal adhesions, microfilaments, and dynamic membrane regions. Together with our recent identification of VASP as a natural ligand of the profilin poly-(L-proline) binding site, our present results suggest that, by linking profilin to zyxin/p83, VASP may participate in spatially confined profilin-regulated F-actin formation.
Reinhard
M
M
Medizinische Universitätsklinik, Klinische Biochemie und Pathobiochemie, Würzburg, Federal Republic of Germany.
Jouvenal
K
K
Tripier
D
D
Walter
U
U
D000595:Amino Acid Sequence; D000818:Animals; D030161:Avian Proteins; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism; D001798:Blood Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000302:isolation & purification / Q000378:metabolism*; D002645:Chickens; D003598:Cytoskeletal Proteins; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005895:Gizzard, Avian; D006023:Glycoproteins; D006801:Humans; D008322:Mammals; D008667:Metalloproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure; D060589:Zyxin; D000098662:Vasodilator-Stimulated Phosphoprotein
D030161:Avian Proteins; D001798:Blood Proteins; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D006023:Glycoproteins; D008667:Metalloproteins; D008840:Microfilament Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; C092842:ZYX protein, human; D060589:Zyxin; D000098662:Vasodilator-Stimulated Phosphoprotein