Quantitative kinetic study of the actin-bundling protein L-plastin and of its impact on actin turn-over.
Quantitative kinetic study of the actin-bundling protein L-plastin and of its impact on actin turn-over.
复制标题
DOI：
10.1371/journal.pone.0009210
复制
发表时间：
2010-02-15
期刊：
影响因子：
3.7
通讯作者：
Friederich E
中科院分区：
文献类型：
--
作者：
Al Tanoury Z;Schaffner-Reckinger E;Halavatyi A;Hoffmann C;Moes M;Hadzic E;Catillon M;Yatskou M;Friederich E
最初在来自实体组织的白细胞和癌细胞中检测到，L-纤溶酶/纤溶酶属于肌动蛋白交联剂的大家族，并被认为是许多癌症的标志物。丝氨酸5残基上的L-纤溶酶磷酸化增加了其F-肌动蛋白结合活性，并且是L-纤溶酶介导的细胞侵袭所必需的。为了研究活细胞中L-plastin的动力学和L-plastin Ser 5磷酸化对L-plastin动力学和肌动蛋白翻转的影响，用GFP偶联的WT-L-plastin、Ser 5取代变体（S5/A、S5/E）或肌动蛋白转染猿猴Vero细胞，并通过光漂白后荧光恢复（FRAP）进行分析。FRAP数据进行了探讨，通过数学建模，以估计稳态反应参数。我们表明，在Vero细胞局灶性粘连L-plastin经历了快速的周期协会/解离后的两个结合状态模型。磷酸化的L-磷脂酶原蛋白增加其协会率的两倍，而解离率不受影响。重要的是，L-纤维蛋白酶影响肌动蛋白周转降低肌动蛋白解离速率的四倍，从而增加的F-肌动蛋白的量在局灶性粘连，所有这些影响被Ser 5磷酸化促进。在MCF-7乳腺癌细胞中，佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）处理诱导L-纤维蛋白酶转位到皱褶膜和棘状结构中的从头肌动蛋白聚合位点，并高度增加其Ser 5磷酸化。PKC同工酶的抑制研究和siRNA敲低均表明新型PKC-δ同工酶参与PMA诱导的信号通路，导致L-plastin Ser 5磷酸化。此外，L-纤维蛋白对肌动蛋白动力学调节的贡献通过其与包括coronin的蛋白质复合物的关联得到证实，coronin已知参与该过程。总之，这些研究结果首次定量证明，L-质体有助于微调肌动蛋白的周转，这是一个活动，这是由丝氨酸5磷酸化促进其高亲和力结合到细胞骨架的调节。在癌细胞中，PKC-δ信号通路似乎将L-plastin磷酸化与肌动蛋白聚合和侵袭联系起来。
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.
登录
查看更多内容
影响因子：
4
作者：
Chen, Chien-Lin;Hsieh, Yeun-Ting;Chen, Hong-Chen
通讯作者：
Chen, Hong-Chen
影响因子：
4.8
作者：
Jones, SL;Brown, EJ
通讯作者：
Brown, EJ
影响因子：
4.8
作者：
Friederich, E;Vancompernolle, K;Vandekerckhove, J
通讯作者：
Vandekerckhove, J
DOI：
10.1083/jcb.127.6.1995
发表时间：
1994-12
期刊：
The Journal of cell biology
影响因子：
--
作者：
Arpin M;Friederich E;Algrain M;Vernel F;Louvard D
通讯作者：
Louvard D
影响因子：
5.6
作者：
Hiroyama, M;Exton, JH
通讯作者：
Exton, JH