The Hedgehog (Hh) receptor Patched1 (PTCH1) is a well-known tumor suppressor that in its active form represses Smoothened (SMO) activity, inhibits proliferation, and induces apoptosis. The cytoplasmic C-terminal domain (CTD) regulates PTCH1 turnover and nucleates a pro-apoptotic complex. In this study, it was mechanistically determined that Autophagy Related 10 (ATG101), essential for mammalian autophagy, physically interacts with the CTD of PTCH1 and connects it to the ULK complex, which stimulates the autophagy machinery in response to changes in nutrient availability. This interaction results in a blockade of basal autophagic flux and accumulation of autophagosomes with undegraded cargo. Remarkably, this function of PTCH1 is independent of its repressive activity on SMO, as shown in SMO-deficient cells or in the presence of a SMO inhibitor, but is opposed by Sonic Hedgehog (SHH). These findings reveal a novel non-canonical function of PTCH1 that limits autophagy, mediated by ATG101, which could have therapeutic implications in Hh-dependent cancers.
The Hedgehog (Hh) receptor PTCH1 and the integral membrane protein 2A (ITM2A) inhibit autophagy by reducing autolysosome formation. In this study, we demonstrate that ITM2A physically interacts with PTCH1; however, the two proteins inhibit autophagic flux independently, since silencing of ITM2A did not prevent the accumulation of LC3BII and p62 in PTCH1-overexpressing cells, suggesting that they provide alternative modes to limit autophagy. Knockdown of ITM2A potentiated PTCH1-induced autophagic flux blockade and increased PTCH1 expression, while ITM2A overexpression reduced PTCH1 protein levels, indicating that it is a negative regulator of PTCH1 non-canonical signalling. Our study also revealed that endogenous ITM2A is necessary for timely induction of myogenic differentiation markers in C2C12 cells since partial knockdown delays the timing of differentiation. We also found that basal autophagic flux decreases during myogenic differentiation at the same time that ITM2A expression increases. Given that canonical Hh signalling prevents myogenic differentiation, we investigated the effect of ITM2A on canonical Hh signalling using GLI-luciferase assays. Our findings demonstrate that ITM2A is a strong negative regulator of GLI transcriptional activity and of GLI1 stability. In summary, ITM2A negatively regulates canonical and non-canonical Hh signalling.
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