Molecular mapping of tyrosine-phosphorylated proteins in focal adhesions using fluorescence resonance energy transfer
Molecular mapping of tyrosine-phosphorylated proteins in focal adhesions using fluorescence resonance energy transfer
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DOI：
10.1242/jcs.02794
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发表时间：
2006-03-01
影响因子：
4
通讯作者：
Geiger, B
中科院分区：
文献类型：
--
作者：
Ballestrem, C;Erez, N;Geiger, B
基于显微镜的荧光共振能量转移（FRET）为监测活细胞自然环境中的分子过程提供了机会。在此我们研究了黏着斑中桩蛋白（paxillin）、Crk相关底物（CAS）和黏着斑激酶（FAK）的分子相互作用以及酪氨酸磷酸化。为此，将这些与青色或黄色荧光蛋白（CFP或YFP）融合的黏着斑磷蛋白在培养的成纤维细胞中表达。为了评估酪氨酸磷酸化的动态过程，我们使用了标记有YFP或CFP的pp60(src)的SH2结构域（dSH2），它能特异性结合磷酸酪氨酸残基。FRET测量结合磷酸特异性抗体的免疫标记显示，FAK、CAS和桩蛋白在早期的基质黏附中发生酪氨酸磷酸化，并且在黏着斑复合物和黏着斑中FAK与CAS和桩蛋白在FRET上接近。数据表明，桩蛋白掺入新生的黏着斑复合物先于其酪氨酸磷酸化，然后磷酸化程度逐渐增加。在用Rho激酶抑制剂处理的细胞或表达组成型活性Rac的细胞中，黏着斑复合物显示出与成熟黏着斑中所见相似的桩蛋白酪氨酸磷酸化水平。基于动态FRET的检测表明，桩蛋白磷酸化发生在黏着斑内的特定区域（热点），而FAK磷酸化则广泛分布。
Microscopy-based fluorescence resonance energy transfer (FRET) provides an opportunity to monitor molecular processes in the natural environment in live cells. Here we studied molecular interactions and tyrosine phosphorylation of paxillin, Crk-associated substrate (CAS), and focal adhesion kinase (FAK) in focal adhesions. For that purpose, these focal adhesion phosphoproteins, fused to cyan or yellow fluorescent proteins (CFP or YFP) were expressed in cultured fibroblasts. To assess the dynamics of tyrosine phosphorylation we used YFP- or CFP-tagged SH2 domain of pp60(src) (dSH2), which specifically binds to phosphotyrosine residues. FRET measurements, combined with immunolabeling with phosphospecific antibodies revealed that FAK, CAS and paxillin are tyrosine phosphorylated in early matrix adhesions and that FAK is in FRET proximity to CAS and paxillin in focal complexes and focal adhesions. Data suggest that paxillin incorporation into nascent focal complexes precedes its tyrosine phosphorylation, which then gradually increases. In cells treated with Rho-kinase inhibitors or expressing constitutively active Rac, focal complexes showed similar levels of paxillin tyrosine phosphorylation as seen in mature focal adhesions. Dynamic FRET-based examination indicated that paxillin phosphorylation occurs in specific areas (hotspots) within focal adhesions, whereas FAK phosphorylation is broadly distributed.