Abstract
The phosphatidylinositol 3-kinase-mammalian target of rapamycin (PI3K-mTOR) pathway plays pivotal roles in cell survival, growth, and proliferation downstream of growth factors. Its perturbations are associated with cancer progression, type 2 diabetes, and neurological disorders. To better understand the mechanisms of action and regulation of this pathway, we initiated a large scale yeast two-hybrid screen for 33 components of the PI3K-mTOR pathway. Identification of 67 new interactions was followed by validation by co-affinity purification and exhaustive literature curation of existing information. We provide a nearly complete, functionally annotated interactome of 802 interactions for the PI3K-mTOR pathway. Our screen revealed a predominant place for glycogen synthase kinase-3 (GSK3) A and B and the AMP-activated protein kinase. In particular, we identified the deformed epidermal autoregulatory factor-1 (DEAF1) transcription factor as an interactor and in vitro substrate of GSK3A and GSK3B. Moreover, GSK3 inhibitors increased DEAF1 transcriptional activity on the 5-HT1A serotonin receptor promoter. We propose that DEAF1 may represent a therapeutic target of lithium and other GSK3 inhibitors used in bipolar disease and depression.
Highlights
The phosphatidylinositol 3-kinase-mammalian target of rapamycin (PI3K-mTOR) pathway plays pivotal roles in cell survival, growth, and proliferation downstream of growth factors
Downstream of AKT, the mammalian target of rapamycin kinase is an essential activator of protein synthesis, promoting cell growth and proliferation [1, 3, 4]. mTOR is regulated by growth factors through AKT, by energy availability through the AMP-activated kinase (AMPK), and by amino acid content through class III phosphatidylinositol 3-kinases (PI3Ks)
They are schematized as part of a receptor module (IGF1, IGF1 receptor, insulin receptor, IRS1, IRS2, and IRS4), a PI3K module
Summary
Yeast Two-hybrid (Y2H) Screens, and Co-affinity Purification (Co-AP) Experiments. Each well of 6-well plates was lysed with 250 l of IP buffer 1 (300 mM NaCl, 50 mM Tris, pH 7.4, 0.5 mM EDTA, 1% Triton X-100, Complete protease inhibitor mixture (Roche Applied Science)) for 30 min on ice. Lysates were sonicated and precleared by centrifugation for 10 min at 14,000 rpm at 4 °C. Beads were resuspended in 1ϫ kinase buffer (25 mM Hepes, pH 7.4, 10 mM MgCl2), and 10 l of beads suspensions were run on a polyacrylamide-SDS gel, transferred to PVDF membranes, and analyzed by Western blotting with anti-Myc (see Fig. 6C) and -M2 (see Fig. 6B, right gel) antibodies to estimate the quantity of Myc-DEAF1 and M2-MLK3 proteins. The identity of RT-PCR products for Deaf from RN46A and 293T cells was assessed by sequencing
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