Analysis of the alpha-actinin/zyxin interaction.
The yeast two-hybrid system was used to search for interaction partners of human zyxin. Screening of two different cDNA libraries, one prepared from human placenta, the other from human heart, yielded several positive clones that occurred in both searches, including clones coding for cyclophilin, nebulette, and alpha-actinin. The zyxin/alpha-actinin interaction was analyzed in detail. By site-directed mutagenesis, a linear motif of 6 amino acids (Phe-Gly-Pro-Val-Val-Ala) present at the N terminus of zyxin was found to play a critical role. Replacement of a single amino acid within this motif abolished binding to alpha-actinin in blot overlays as well as in living cells. On the other hand, the interaction site in alpha-actinin was mapped to a conformational determinant present in the center of the protein as demonstrated by a fragment deletion analysis. This binding site involved a tandem array of two complete spectrin-like domains. Only fragments that were able to dimerize in yeast also bound to zyxin, suggesting that dimerization of alpha-actinin is essential for zyxin binding.
Li
B
B
M. E. Müller Institute, University of Bern, P. O. Box 30, CH-3010 Bern, Switzerland.
Trueb
B
B
D000185:Actinin / Q000737:chemistry* / Q000378:metabolism*; D020816:Amino Acid Motifs; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002478:Cells, Cultured; D002642:Chick Embryo; D003598:Cytoskeletal Proteins; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D005347:Fibroblasts / Q000378:metabolism; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D006023:Glycoproteins; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009206:Myocardium / Q000378:metabolism; D010920:Placenta / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D015533:Transcriptional Activation; D014162:Transfection; D020798:Two-Hybrid System Techniques; D060589:Zyxin
D000596:Amino Acids; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D006023:Glycoproteins; D008164:Luminescent Proteins; D008667:Metalloproteins; C092842:ZYX protein, human; D060589:Zyxin; D000185:Actinin; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase