Abstract
Zyxin contains a proline-rich N-terminal domain that is similar to the C-terminal domain in the ActA protein of the bacteria, Listeria monocytogenes. We screened the entire amino acid sequence of human zyxin for Mena-interacting peptides and found that, as with ActA, proline-rich sequences were the sole zyxin sequences capable of binding to Ena/vasodilator-stimulated phosphoprotein (VASP) family members in vitro. From this information, we tested zyxin mutants in which the proline-rich sequences were altered. The reduction in Mena/VASP binding was confirmed by peptide tests, immunoprecipitation, and ectopic expression of zyxin variants at the surface of mitochondria. By transfection assays we showed that zyxin interaction with Mena/VASP in vivo enhances the production of actin-rich structures at the apical surface of cells. Microinjection into cells of peptides corresponding to the first proline-rich sequence of zyxin caused the loss of Mena/VASP from focal contacts. Furthermore, these peptides reduced the degree of spreading of cells replated after trypsinization. We conclude that zyxin and proteins that harbor similar proline-rich repeats contribute to the positioning of Mena/VASP proteins. The positioning of Ena/VASP family members appears to be important when the actin cytoskeleton is reorganized, such as during spreading.
Highlights
Introduction of aZyxin-derived Proline-rich Peptide into Cells Causes Mislocalization of Ena/VASP Members and Reduces the Degree of Cell Spreading on Fibronectin—As described above, comparison of the effects of membrane-targeted Nt-zyxin and the Nt(F71A/F93A/F104A/F114A) mutant form of zyxin revealed a role for Ena/VASP binding capacity in the zyxin-dependent changes in actin assembly and organization that we observe by employing the CAAX assay
The EVH1 Domain of Mena Interacts with Proline-rich Sequences in the N-terminal Region of Human Zyxin—We were interested in probing the physiological significance of the ability of zyxin to bind Ena/VASP proteins
Since our goal was to compromise the ability of zyxin to bind Ena/VASP family of proteins and to explore the physiological consequences of this interaction, we precisely mapped the zyxin amino acid sequences capable of binding GST-Mena(6 –170) using a SPOTs custom-synthesized peptide library of 187 peptides that spanned the entire human zyxin sequence
Summary
Introduction of aZyxin-derived Proline-rich Peptide into Cells Causes Mislocalization of Ena/VASP Members and Reduces the Degree of Cell Spreading on Fibronectin—As described above, comparison of the effects of membrane-targeted Nt-zyxin and the Nt(F71A/F93A/F104A/F114A) mutant form of zyxin revealed a role for Ena/VASP binding capacity in the zyxin-dependent changes in actin assembly and organization that we observe by employing the CAAX assay. We have utilized a zyxin-derived peptide, Zyx-(67– 81)-(PPEDFPLPPPPLAGD), which corresponds to the amino acid sequence of the first proline-rich repeat (spot A23, Fig. 1A) and primary binding site for Ena/ VASP family members in zyxin, to inhibit competitively the ability of zyxin to dock these proteins in vivo. Introduction of the Zyx-(67– 81)-(F71A) peptide had no apparent effect on Mena localization (Fig. 7, E and F). These results further confirm that the Ena/VASP interaction with zyxin detected in vitro likely occurs in vivo and is important for the normal subcellular distribution of Ena/VASP family of proteins
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
