Saitou Takashi, Kajiwara Kentaro, Oneyama Chitose, Suzuki Takashi, Okada Masato
Department of Molecular Medicine for Pathogenesis, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime, Japan.
Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
PLoS One. 2014 Mar 27;9(3):e93470. doi: 10.1371/journal.pone.0093470. eCollection 2014.
The tyrosine kinase c-Src is upregulated in numerous human cancers, implying a role for c-Src in cancer progression. Previously, we have shown that sequestration of activated c-Src into lipid rafts via a transmembrane adaptor, Cbp/PAG1, efficiently suppresses c-Src-induced cell transformation in Csk-deficient cells, suggesting that the transforming activity of c-Src is spatially regulated via Cbp in lipid rafts. To dissect the molecular mechanisms of the Cbp-mediated regulation of c-Src, a combined analysis was performed that included mathematical modeling and in vitro experiments in a c-Src- or Cbp-inducible system. c-Src activity was first determined as a function of c-Src or Cbp levels, using focal adhesion kinase (FAK) as a crucial c-Src substrate. Based on these experimental data, two mathematical models were constructed, the sequestration model and the ternary model. The computational analysis showed that both models supported our proposal that raft localization of Cbp is crucial for the suppression of c-Src function, but the ternary model, which includes a ternary complex consisting of Cbp, c-Src, and FAK, also predicted that c-Src function is dependent on the lipid-raft volume. Experimental analysis revealed that c-Src activity is elevated when lipid rafts are disrupted and the ternary complex forms in non-raft membranes, indicating that the ternary model accurately represents the system. Moreover, the ternary model predicted that, if Cbp enhances the interaction between c-Src and FAK, Cbp could promote c-Src function when lipid rafts are disrupted. These findings underscore the crucial role of lipid rafts in the Cbp-mediated negative regulation of c-Src-transforming activity, and explain the positive role of Cbp in c-Src regulation under particular conditions where lipid rafts are perturbed.
酪氨酸激酶c-Src在多种人类癌症中上调，这意味着c-Src在癌症进展中发挥作用。此前，我们已经表明，通过跨膜衔接蛋白Cbp/PAG1将活化的c-Src隔离到脂筏中，可有效抑制Csk缺陷细胞中c-Src诱导的细胞转化，这表明c-Src的转化活性通过脂筏中的Cbp进行空间调节。为了剖析Cbp介导的c-Src调节的分子机制，我们进行了一项综合分析，包括数学建模和在c-Src或Cbp诱导系统中的体外实验。首先，以粘着斑激酶（FAK）作为关键的c-Src底物，确定c-Src活性是c-Src或Cbp水平的函数。基于这些实验数据，构建了两个数学模型，即隔离模型和三元模型。计算分析表明，这两个模型都支持我们的观点，即Cbp的脂筏定位对于抑制c-Src功能至关重要，但包含由Cbp、c-Src和FAK组成的三元复合物的三元模型还预测，c-Src功能取决于脂筏体积。实验分析表明，当脂筏被破坏且三元复合物在非脂筏膜中形成时，c-Src活性会升高，这表明三元模型准确地反映了该系统。此外，三元模型预测，如果Cbp增强c-Src与FAK之间的相互作用，那么在脂筏被破坏的特定条件下，Cbp可能会促进c-Src功能。这些发现强调了脂筏在Cbp介导的c-Src转化活性负调节中的关键作用，并解释了在脂筏受到干扰的特定条件下Cbp在c-Src调节中的积极作用。
