Abstract
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.
Highlights
The first identified proto-oncogene product, c-Src [1], is a membrane-associated non-receptor tyrosine kinase that plays pivotal roles in coordinating a broad range of cellular responses such as differentiation, proliferation, adhesion, and migration [2]
This study addressed molecular mechanisms of the spatial regulation of c-Src by combining mathematical modeling and in vitro experimental approaches
Both models support our proposal that raft localization of Csk-binding protein (Cbp) is crucial for suppression of c-Src function, but the ternary model predicts the dependence of c-Src function on the lipid-raft volume
Summary
The first identified proto-oncogene product, c-Src [1], is a membrane-associated non-receptor tyrosine kinase that plays pivotal roles in coordinating a broad range of cellular responses such as differentiation, proliferation, adhesion, and migration [2]. C-Src is frequently overexpressed and activated in a wide variety of human cancers [6,11,12], despite the fact that Csk is normally expressed and the c-src gene is not mutated [13,14]. These observations suggest that other components in the c-Src regulatory system may be perturbed during cancer progression, the underlying mechanisms remain unclear. Upon activation of c-Src, c-Src and FAK tightly interact to phosphorylate and activate each other, but the mechanism through which activated c-Src efficiently accesses FAK remains elusive
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