Wei Zhang1, Suat Peng Neo1, Jayantha Gunaratne1, Anders Poulsen2, Liu Boping2,Esther Hongqian Ong21, Kanda Sangthongpitag2, Vishal Pendharkar2, Jeffrey Hill2 & Stephen M Cohen1,3
1 Institute of Molecular and Cell Biology,
61 Biopolis Drive, Singapore 138673
2 Experimental Therapeutics Center,
31 Biopolis Way, Singapore 138669
3 Department of Biological Sciences, National University of Singapore117543
Published online ahead of print in Cellular Signalling on 19 December 2014.
The high proliferation rate of cancer cells, together with environmental factors such as hypoxia and nutrient deprivation can cause Endoplasmic Reticulum (ER) stress. The protein kinase PERK is an essential mediator in one of three ER stress response pathways. Genetic and pharmacological inhibition of PERK has been reported to limit tumor growth in xenograft models. Here we provide evidence that inactive PERK interacts with the nuclear pore associated Vault complex proteins, compromising Vaults-mediated nuclear transport of PTEN. As a result, pharmacological inhibition of PERK under ER stress shows an abnormal sequestration of the Vault complex by high level of inactive PERK, leading to feedback regulation of the PI3K/AKT pathway through increased cytoplasmic PTEN activity. As the PI3K/PTEN/AKT pathway is crucial for many aspects of cell growth and survival, this unexpected effect of PERK inhibitors to lower AKT activity may have implications for the potential use of PERK inhibitors as therapeutic agents.
Figure: Inactive PERK interacts with Vault complex.
Figure Legend: Systematic Survey of miRNA Mutant Phenotypes
(A) SILAC pull-down workflow. H1299 cells were transfected to express PERKK622M-Flag or intact PERK-Flag and grown in Heavy vs Light SILAC media. Flag immunoprecipitation was used to recover PERK and bound proteins for mass spectometric analysis.
(B) Ratio-intensity plot. The red data points indicate proteins that preferentially interacted with PERKK622Mvs native PERK with ap value (Ratio Significance) < 0.05.
(C) Diagram of the Vault complex modified from (4). The capsid structure consists of MVP molecules (red). Gray circles indicate PARP4. vRNA (empty circles) at the end of cap region co-localizes with TEP1 (blue).
(D) Immunoblots to monitor the interaction between MVP-GFP and PERK or PERKK622M. H1299 cells were transfected to express the indicated proteins. GFP-tagged MVP was recovered using GFP-trap beads, and blots were probed with anti-GFP and anti-PERK. Input shows 1% of the lysate. IP and input sets with active and inactive PERK were run on the same gel but shown separately.
(E) Immunoblots to monitor the interaction between MVP-GFP and PERK as in D. H1299 cells were treated with cpd2, as indicated. Input shows 1% of the lysate. IP and input sets with active PERK treated with or without compound 2 were run on the same gel but shown separately.
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