YAP1 is amplified and up-regulated in hedgehog-associated medulloblastomas and mediates Sonic hedgehog-driven neural precursor proliferation

Africa Fernandez-L; Paul A. Northcott; James Dalton; Charles Fraga; David Ellison; Stephane Angers; Michael D. Taylor; Anna Marie Kenney

doi:10.1101/gad.1824509

Figure 3.

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IRS1 interacts with YAP1 and regulates its nuclear accumulation. (A) Subcellular fractionation of CGNPs cultured in the presence or absence of Shh. (Left) The greatest accumulation of IRS1 protein after Shh treatment takes place in the nucleus. (Right) Immunoprecipitation of IRS1 in Shh-treated CGNPs brings down YAP1. (B, top panel, left) Immunostaining shows that YAP1 is mainly nuclear in Shh-treated CGNPs. (Top panel, second from left) In the presence of cyclopamine, YAP1 is excluded from the nucleus. (Top panel, second from right) In the presence of cyclopamine and leptomycin, YAP1 accumulates in the nucleus. Overexpressing IRS1 in the presence of cyclopamine leads to YAP1 accumulation in the nucleus (bottom panel, left), while overexpressing control viruses (GFP) does not (top panel, right). (Bottom panel, second from left) Cells that get infected with IRS1 (GFP reporter) viruses have nuclear YAP1. (Bottom panel, second from right) IRS1 knockdown prevents YAP1 from accumulating in the nucleus. (Bottom panel, right) The knockdown efficiency is shown by Western blot. (C) Immunoprecipitation of IRS1 in the Pzp53 medulloblastoma cell line coprecipitates YAP1. (Left) The YAP1:IRS1 interaction was decreased in the presence of cyclopamine. (Right) IRS1 was also detected in YAP1 immunoprecipitates. (D) Subcellular fractionation of Pzp53 cells and subsequent IRS1 immunoprecipitation. YAP1 was detected in both the nuclear and cytoplasmic precipitates. (E) Pzp53med cells were treated with leptomycin, fixed, and immunostained for YAP1 and IRS1. (Top row) In untreated cells, YAP1 and IRS1 are both nuclear and cytoplasmic. (Bottom row) In leptomycin-treated cells, there is an accumulation of both proteins in the nucleus. (F) Immunoprecipitation of CRM1 in Pzp53 cells. Both YAP1 and IRS1 were detected in the immunoprecipitate.