Article (Scientific journals)
Quantitative kinetic study of the actin-bundling protein L-plastin and of its impact on actin turn-over.
Al Tanoury, Ziad; Schaffner-Reckinger, Elisabeth; Halavatyi, Aliaksandr et al.
2010In PLoS ONE, 5 (2), p. 9210
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Keywords :
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
Abstract :
[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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Al Tanoury, Ziad
Schaffner-Reckinger, Elisabeth ;  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 ;  University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit
Hadzic, Ermin ;  University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit
Catillon, Marie ;  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
External co-authors :
no
Language :
English
Title :
Quantitative kinetic study of the actin-bundling protein L-plastin and of its impact on actin turn-over.
Publication date :
2010
Journal title :
PLoS ONE
ISSN :
1932-6203
Publisher :
Public Library of Science, United States - California
Volume :
5
Issue :
2
Pages :
e9210
Peer reviewed :
Peer Reviewed verified by ORBi
Available on ORBilu :
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