Specific tyrosine phosphorylation sites on cortactin regulate Nck1-dependent actin polymerization in invadopodia
Specific tyrosine phosphorylation sites on cortactin regulate Nck1-dependent actin polymerization in invadopodia
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DOI：
10.1242/jcs.068163
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发表时间：
2010-11-01
影响因子：
4
通讯作者：
Condeelis, John
中科院分区：
文献类型：
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作者：
Oser, Matthew;Mader, Christopher C.;Condeelis, John
浸润状突起是侵袭性癌细胞中基质降解膜突起，富含调节肌动蛋白聚合的蛋白质。在invadopdia中，启动肌动蛋白聚合的开关需要酪氨酸残基421、466和482在接触上的磷酸化。然而，目前尚不清楚这些接触酪氨酸磷酸化位点中哪一个控制肌动蛋白聚合。我们研究了单独的酪氨酸磷酸化位点（421、466和482）在接触中对肌动蛋白聚合的调节作用。我们提供的证据表明，酪氨酸421和466的磷酸化，而不是482，是在侵殖虫中产生游离肌动蛋白倒刺末端所必需的。此外，这些相同的磷酸酪氨酸对于Nck1通过SH2结构域募集到invadopodia，对于Nck1在体外与接触的直接结合，以及在invadopodia中Nck1与接触之间的FRET相互作用是重要的。此外，在表达磷酸酪氨酸421或466突变的细胞中，基质蛋白水解依赖的肿瘤细胞侵袭被显著抑制。总之，这些结果确定了酪氨酸421和466在接触蛋白上的磷酸化是调节nck1依赖性肌动蛋白聚合和肿瘤细胞侵袭的关键残基，并表明特异性阻断酪氨酸421或466磷酸化可能有效抑制肿瘤细胞在体内的侵袭。
Invadopodia are matrix-degrading membrane protrusions in invasive carcinoma cells enriched in proteins that regulate actin polymerization. The on-off regulatory switch that initiates actin polymerization in invadopodia requires phosphorylation of tyrosine residues 421, 466, and 482 on cortactin. However, it is unknown which of these cortactin tyrosine phosphorylation sites control actin polymerization. We investigated the contribution of individual tyrosine phosphorylation sites (421, 466, and 482) on cortactin to the regulation of actin polymerization in invadopodia. We provide evidence that the phosphorylation of tyrosines 421 and 466, but not 482, is required for the generation of free actin barbed ends in invadopodia. In addition, these same phosphotyrosines are important for Nck1 recruitment to invadopodia via its SH2 domain, for the direct binding of Nck1 to cortactin in vitro, and for the FRET interaction between Nck1 and cortactin in invadopodia. Furthermore, matrix proteolysis-dependent tumor cell invasion is dramatically inhibited in cells expressing a mutation in phosphotyrosine 421 or 466. Together, these results identify phosphorylation of tyrosines 421 and 466 on cortactin as the crucial residues that regulate Nck1-dependent actin polymerization in invadopodia and tumor cell invasion, and suggest that specifically blocking either tyrosine 421 or 466 phosphorylation might be effective at inhibiting tumor cell invasion in vivo.