Reference : Quantitative kinetic study of the actin-bundling protein L-plastin and of its impact ...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/10993/10716
Quantitative kinetic study of the actin-bundling protein L-plastin and of its impact on actin turn-over.
English
Al Tanoury, Ziad [> >]
Schaffner-Reckinger, Elisabeth mailto [University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit >]
Halavatyi, Aliaksandr [University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit >]
Hoffmann, Celine [> >]
Moes, Michèle mailto [University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit >]
Hadzic, Ermin mailto [University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit >]
Catillon, Marie mailto [University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit >]
Yatskou, Mikalai [> >]
Friederich, Evelyne [University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit >]
2010
PloS one
5
2
e9210
Yes (verified by ORBilu)
International
1932-6203
1932-6203
United States
[en] Actins/metabolism ; Algorithms ; Amino Acid Substitution ; Animals ; Cell Line, Tumor ; Cercopithecus aethiops ; Cortactin/metabolism ; Cytoskeleton/metabolism ; Fluorescence Recovery After Photobleaching ; Focal Adhesions/metabolism ; Green Fluorescent Proteins/genetics/metabolism ; Humans ; Kinetics ; Membrane Glycoproteins/genetics/metabolism ; Microfilament Proteins/genetics/metabolism ; Models, Biological ; Phosphorylation/drug effects ; Protein Binding ; Protein Kinase C-delta/genetics/metabolism ; Protein Transport/drug effects ; RNA Interference ; Recombinant Fusion Proteins/genetics/metabolism ; Serine/genetics/metabolism ; Tetradecanoylphorbol Acetate/pharmacology ; Transfection ; Vero Cells
[en] BACKGROUND: Initially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers. Phosphorylation of L-plastin on residue Ser5 increases its F-actin binding activity and is required for L-plastin-mediated cell invasion. METHODOLOGY/PRINCIPAL FINDINGS: To study the kinetics of L-plastin and the impact of L-plastin Ser5 phosphorylation on L-plastin dynamics and actin turn-over in live cells, simian Vero cells were transfected with GFP-coupled WT-L-plastin, Ser5 substitution variants (S5/A, S5/E) or actin and analyzed by fluorescence recovery after photobleaching (FRAP). FRAP data were explored by mathematical modeling to estimate steady-state reaction parameters. We demonstrate that in Vero cell focal adhesions L-plastin undergoes rapid cycles of association/dissociation following a two-binding-state model. Phosphorylation of L-plastin increased its association rates by two-fold, whereas dissociation rates were unaffected. Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation. In MCF-7 breast carcinoma cells, phorbol 12-myristate 13-acetate (PMA) treatment induced L-plastin translocation to de novo actin polymerization sites in ruffling membranes and spike-like structures and highly increased its Ser5 phosphorylation. Both inhibition studies and siRNA knock-down of PKC isozymes pointed to the involvement of the novel PKC-delta isozyme in the PMA-elicited signaling pathway leading to L-plastin Ser5 phosphorylation. Furthermore, the L-plastin contribution to actin dynamics regulation was substantiated by its association with a protein complex comprising cortactin, which is known to be involved in this process. CONCLUSIONS/SIGNIFICANCE: Altogether these findings quantitatively demonstrate for the first time that L-plastin contributes to the fine-tuning of actin turn-over, an activity which is regulated by Ser5 phosphorylation promoting its high affinity binding to the cytoskeleton. In carcinoma cells, PKC-delta signaling pathways appear to link L-plastin phosphorylation to actin polymerization and invasion.
http://hdl.handle.net/10993/10716
10.1371/journal.pone.0009210

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