Quantitative kinetic study of the actin-bundling protein L-plastin and of its impact on actin turn-over.
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.


RESULTS
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
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.
Al Tanoury
Ziad
Z
Laboratory of Cytoskeleton and Cell Plasticity, Life Sciences Research Unit, University of Luxembourg, Luxembourg City, Luxembourg.
Schaffner-Reckinger
Elisabeth
E
Halavatyi
Aliaksandr
A
Hoffmann
Céline
C
Moes
Michèle
M
Hadzic
Ermin
E
Catillon
Marie
M
Yatskou
Mikalai
M
Friederich
Evelyne
E
D000199:Actins / Q000378:metabolism*; D000465:Algorithms; D019943:Amino Acid Substitution; D000818:Animals; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D051356:Cortactin / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D022001:Focal Adhesions / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D051745:Protein Kinase C-delta / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014709:Vero Cells
D000199:Actins; D051356:Cortactin; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; C026742:plastin; D049452:Green Fluorescent Proteins; D012694:Serine; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate