References of "Beckerle, M. C."
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See detailTargeting of zyxin to sites of actin membrane interaction and to the nucleus.
Nix, D. A.; Fradelizi, J.; Bockholt, S. et al

in The Journal of biological chemistry (2001), 276(37), 34759-67

The localization of proteins to particular intracellular compartments often regulates their functions. Zyxin is a LIM protein found prominently at sites of cell adhesion, faintly in leading lamellipodia ... [more ▼]

The localization of proteins to particular intracellular compartments often regulates their functions. Zyxin is a LIM protein found prominently at sites of cell adhesion, faintly in leading lamellipodia, and transiently in cell nuclei. Here we have performed a domain analysis to identify regions in zyxin that are responsible for targeting it to different subcellular locations. The N-terminal proline-rich region of zyxin, which harbors binding sites for alpha-actinin and members of the Ena/VASP family, concentrates in lamellipodial extensions and weakly in focal adhesions. The LIM region of zyxin displays robust targeting to focal adhesions. When overexpressed in cells, the LIM region of zyxin causes displacement of endogenous zyxin from focal adhesions. Upon mislocalization of full-length zyxin, at least one member of the Ena/VASP family is also displaced, and the organization of the actin cytoskeleton is perturbed. Zyxin also has the capacity to shuttle between the nucleus and focal adhesion sites. When nuclear export is inhibited, zyxin accumulates in cell nuclei. The nuclear accumulation of zyxin occurs asynchronously with approximately half of the cells exhibiting nuclear localization of zyxin within 2.3 h of initiating leptomycin B treatment. Our results provide insight into the functions of different zyxin domains. [less ▲]

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See detailCharacterization of the interaction between zyxin and members of the Ena/vasodilator-stimulated phosphoprotein family of proteins.
Drees, B.; Friederich, Evelyne UL; Fradelizi, J. et al

in The Journal of biological chemistry (2000), 275(29), 22503-11

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 ... [more ▼]

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. [less ▲]

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See detailQuantitative measurement of proteins by western blotting with Cy5-coupled secondary antibodies.
Fradelizi, J.; Friederich, Evelyne UL; Beckerle, M. C. et al

in BioTechniques (1999), 26(3), 484-6488490

The concentration of proteins in cells is an important parameter that determines how a protein will interact with other proteins or pharmacological agents. Recent developments in Western blotting ... [more ▼]

The concentration of proteins in cells is an important parameter that determines how a protein will interact with other proteins or pharmacological agents. Recent developments in Western blotting techniques have now made this a method of choice to measure protein concentration in complex solutions such as total cell extracts. We show that detection of Cy5-coupled secondary antibodies by PhosphorImager analysis produces signals that approach linearity with respect to protein concentration over a 20-fold range. We used this technique to estimate cellular levels of zyxin, which is an important protein component of the actin cytoskeleton in mammalian cells. By producing specific protein standards based on sequences that are available from public databases, it is now possible to estimate the concentration of almost any protein by this technique. [less ▲]

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See detailStructural and functional similarities between the human cytoskeletal protein zyxin and the ActA protein of Listeria monocytogenes.
Golsteyn, R. M.; Beckerle, M. C.; Koay, T. et al

in Journal of cell science (1997), 110 ( Pt 16)

The intracellular bacterial parasite Listeria monocytogenes produces ActA protein at its surface to facilitate the localized assembly of actin-filled comets that are required for movement. The ... [more ▼]

The intracellular bacterial parasite Listeria monocytogenes produces ActA protein at its surface to facilitate the localized assembly of actin-filled comets that are required for movement. The organization of actin in Listeria comets shows striking similarity to the organization of actin at the plasma membrane of mammalian cells. Therefore we examined the possibility that an ActA-like protein is present in mammalian cells. By using antibodies directed against ActA, we identified zyxin as an ActA related protein in a number of cell types. We compared the functions of ActA and zyxin by transient expression of variants tagged with an inner plasma membrane localization sequence (a CAAX box). Targeting of the proline rich domain of zyxin to the plasma membrane disrupts the actin cytoskeleton and cell shape in a manner similar to that which occurs with membrane-targeted ActA sequences. A chimeric protein composed of the N-terminal domain of ActA fused to the N-terminal and central domains of zyxin induced a full ActA response in cells. Furthermore, zyxin and ActA exhibit common protein partners in vitro. On the basis of the shared properties of zyxin and ActA, we propose that zyxin enhances actin organizing activity in mammalian cells. [less ▲]

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