Abstract
Feedback control is a key mechanism in signal transduction, intimately involved in regulating the outcome of the cellular response. Here, we report a novel mechanism by which PHLDA1, Pleckstrin homology-like domain, family A, member 1, negatively regulates ErbB receptor signaling by inhibition of receptor oligomerization. We have found that the ErbB3 ligand, heregulin, induces PHILDA1 expression in MCF-7 cells. Transcriptionally-induced PHLDA1 protein directly binds to ErbB3, whereas knockdown of PHLDA1 increases complex formation between ErbB3 and ErbB2. To provide insight into the mechanism for our time-course and single-cell experimental observations, we performed a systematic computational search of network topologies of the mathematical models based on receptor dimer-tetramer formation in the ErbB activation processes. Our results indicate that only a model in which PHLDA1 inhibits formation of both dimers and tetramer can explain the experimental data. Predictions made from this model were further validated by single-molecule imaging experiments. Our studies suggest a unique regulatory feature of PHLDA1 to inhibit the ErbB receptor oligomerization process and thereby control the activity of receptor signaling network.
Highlights
U0126 and the Akt inhibitor VIII, a specific inhibitor targeting Akt1 and -2, decreased the induction of PHLDA1 mRNA at 2 h after HRG stimulation. These results suggest that PHLDA1 mRNA induction is dependent on both Ras-extracellular signal-regulated kinase (ERK) and PI3K-Akt pathways
Among the molecules we analyzed for phosphorylation, ErbB2 was most affected (1.8 times higher than the control), and the phosphorylation of EGFR, ErbB2, and ErbB3 was significantly up-regulated by PHLDA1 knockdown (p Ͻ 0.05, Welch’s statistical test, Fig. S4)
Consistent with the above findings, PHLDA1 overexpression inhibited phosphorylation of ErbB2, Akt, and ERK in the plasma membrane fraction with statistical significance (Fig. 1H and Fig. S5), implying that PHLDA1 is responsible for negative regulation of the ErbB signaling pathway
Summary
Feedback control is a key mechanism in signal transduction, intimately involved in regulating the outcome of the cellular response. We have found using liquid chromatography-mass spectrometry (LC/ MS) that PHLDA1 targets ErbB3 and thereby inhibits phosphorylation of ErbB receptors in HRG-stimulated MCF-7 cells These experimental results suggest a role for PHLDA1 in negative regulation of the receptors, single-cell data have shown that the expression of PHLDA1 and phospho-ErbB2 are positively correlated, even at the time when phosphorylation of ErbB2 is attenuated and PHLDA1 expression is increased. Mathematical models, including ErbB receptor activation processes such as dimerization, phosphorylation, and tetramer formation with different inhibitory modes of PHLDA1, demonstrated that only a model containing inhibition of both dimer and tetramer formation could explain the experimental data. Our study suggests that PHLDA1 inhibits higher-order oligomerization of the ErbB receptor via a transcriptionally-induced feedback mechanism
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