Quantitative Kinetic Study of the Actin-Bundling Protein L-Plastin and of Its Impact on Actin Turn-Over
Open Access
- 15 February 2010
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLOS ONE
- Vol. 5 (2), e9210
- https://doi.org/10.1371/journal.pone.0009210
Abstract
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. 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-δ 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. 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-δ signaling pathways appear to link L-plastin phosphorylation to actin polymerization and invasion.Keywords
This publication has 59 references indexed in Scilit:
- The actin filament cross-linker L-plastin confers resistance to TNF-α in MCF-7 breast cancer cells in a phosphorylation-dependent mannerJournal of Cellular and Molecular Medicine, 2009
- Cortactin Promotes Migration and Platelet-derived Growth Factor-induced Actin Reorganization by Signaling to Rho-GTPasesMolecular Biology of the Cell, 2009
- The rate of N-WASP exchange limits the extent of ARP2/3-complex-dependent actin-based motilityNature, 2009
- Regulation of Actin Assembly Associated With Protrusion and Adhesion in Cell MigrationPhysiological Reviews, 2008
- Suppression of PMA-induced tumor cell invasion by capillarisin via the inhibition of NF-κB-dependent MMP-9 expressionBiochemical and Biophysical Research Communications, 2008
- High-resolution cryo-EM structure of the F-actin–fimbrin/plastin ABD2 complexProceedings of the National Academy of Sciences, 2008
- Role of fascin in filopodial protrusionThe Journal of cell biology, 2006
- Stress fibers are generated by two distinct actin assembly mechanisms in motile cellsThe Journal of cell biology, 2006
- Cortactin: coupling membrane dynamics to cortical actin assemblyOncogene, 2001
- Differential Sensitivity of Breast Cancer Cells to Tumor Necrosis Factor-α: Involvement of Protein Kinase CBiochemical and Biophysical Research Communications, 2001