Laboratory of Cytoskeleton and Cell Plasticity, Life Sciences Research Unit, University of Luxembourg, Luxembourg City, Luxembourg.
PLoS One. 2010 Feb 15;5(2):e9210. doi: 10.1371/journal.pone.0009210.
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.
L-塑丝蛋白/微丝结合蛋白最初在白细胞和来源于实体组织的癌细胞中被检测到，属于肌动蛋白交联蛋白大家族，被认为是许多癌症的标志物。L-塑丝蛋白残基丝氨酸 5 的磷酸化增加了其与 F-肌动蛋白的结合活性，并且对于 L-塑丝蛋白介导的细胞侵袭是必需的。
方法/主要发现：为了研究 L-塑丝蛋白的动力学以及 L-塑丝蛋白丝氨酸 5 磷酸化对活细胞中 L-塑丝蛋白动力学和肌动蛋白周转率的影响，用 GFP 偶联的 WT-L-塑丝蛋白、丝氨酸 5 取代变体（S5/A、S5/E）或肌动蛋白转染猴肾 Vero 细胞，并通过光漂白后荧光恢复（FRAP）进行分析。通过数学建模探索 FRAP 数据，以估计稳态反应参数。我们证明，在 Vero 细胞焦点黏附处，L-塑丝蛋白经历快速的缔合/解离循环，遵循双结合状态模型。L-塑丝蛋白的磷酸化使结合速率增加了两倍，而解离速率不受影响。重要的是，L-塑丝蛋白通过将肌动蛋白的解离速率降低四倍来影响肌动蛋白周转率，从而增加焦点黏附处的 F-肌动蛋白量，所有这些作用都由丝氨酸 5 磷酸化促进。在 MCF-7 乳腺癌细胞中，佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）处理诱导 L-塑丝蛋白易位到皱襞细胞膜和刺状结构中的新肌动蛋白聚合位点，并高度增加其丝氨酸 5 磷酸化。抑制研究和 PKC 同工型的 siRNA 敲低都指向新型 PKC-δ同工型参与 PMA 引发的信号通路，导致 L-塑丝蛋白丝氨酸 5 磷酸化。此外，L-塑丝蛋白与包含 cortactin 的蛋白质复合物的关联证实了其对肌动蛋白动力学调节的贡献，cortactin 已知参与该过程。
结论/意义：总之，这些发现首次定量证明，L-塑丝蛋白有助于肌动蛋白周转率的微调，该活性受丝氨酸 5 磷酸化调节，促进其与细胞骨架的高亲和力结合。在癌细胞中，PKC-δ信号通路似乎将 L-塑丝蛋白磷酸化与肌动蛋白聚合和侵袭联系起来。
