Abstract
BackgroundCell adhesion has been shown to induce activation of certain growth factor receptors in a ligand-independent manner. However, the mechanism for such activation remains obscure.MethodsHuman epidermal carcinoma A431 cells were used as a model to examine the mechanism for adhesion-induced activation of hepatocyte growth factor receptor Met and epidermal growth factor receptor (EGFR). The cells were suspended and replated on culture dishes under various conditions. The phosphorylation of Met at Y1234/1235 and EGFR at Y1173 were used as indicators for their activation. The distribution of the receptors and lipid rafts on the plasma membrane were visualized by confocal fluorescent microscopy and total internal reflection microscopy.ResultsWe demonstrate that Met and EGFR are constitutively activated in A431 cells, which confers proliferative and invasive potentials to the cells. The ligand-independent activation of Met and EGFR in A431 cells relies on cell adhesion to a substratum, but is independent of cell spreading, extracellular matrix proteins, and substratum stiffness. This adhesion-induced activation of Met and EGFR cannot be attributed to Src activation, production of reactive oxygen species, and the integrity of the cytoskeleton. In addition, we demonstrate that Met and EGFR are independently activated upon cell adhesion. However, partial depletion of Met and EGFR prevents their activation upon cell adhesion, suggesting that overexpression of the receptors is a prerequisite for their self-activation upon cell adhesion. Although Met and EGFR are largely distributed in 0.04% Triton-insoluble fractions (i.e. raft fraction), their activated forms are detected mainly in 0.04% Triton-soluble fractions (i.e. non-raft fraction). Upon cell adhesion, lipid rafts are accumulated at the cell surface close to the cell-substratum interface, while Met and EGFR are mostly excluded from the membrane enriched by lipid rafts.ConclusionsOur results suggest for the first time that cell adhesion to a substratum may induce a polarized distribution of lipid rafts to the cell-substratum interface, which may allow Met and EGFR to be released from lipid rafts, thus leading to their activation in a ligand-independent manner.
Highlights
Cell adhesion has been shown to induce activation of certain growth factor receptors in a ligandindependent manner
We found that both Met and epidermal growth factor receptor (EGFR) are constitutively activated in human epidermal A431 cells (Figure 1a)
Met and EGFR are independently activated upon cell adhesion As Met and EGFR have been reported to physically interact with and activate each other [18,19,20], we examined if adhesion-induced activation of Met and EGFR in A431 cells relies on their mutual activation
Summary
Cell adhesion has been shown to induce activation of certain growth factor receptors in a ligandindependent manner. Aberrant activation of receptor tyrosine kinases (RTKs) is one of the major causes for malignant transformation [1]. Cell-matrix adhesion is involved in lipid rafts-mediated signal transduction pathways [11]. Integrin a6b4, a laminin receptor, is incorporated in lipid rafts through palmitoylation at cysteine in the membrane-proximal segment of b4 tail, which subsequently activates a palmitoylated Src family kinase in the rafts, important for mitogenic signalling [12]. It has been demonstrated that integrin-mediated adhesion regulates the trafficking of lipid rafts components. RalA, a small GTPase, was identified as a key determinant for integrin-dependent membrane rafts trafficking and regulation of growth signalling [13]
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