Regulation of cellular growth in adverse environmental conditions

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OBJECTIVE: The Long Term Goal of my NSERC-funded research program is to gain a basic understanding of the underlying mechanisms that regulate cellular proliferation in adverse environments. The overall objectives of this research are to determine the O2 sensing mechanism/s controlled by hypoxia and how they affect IGF-dependent cell proliferation. BACKGROUND: The mechanistic target of rapamycin (mTOR) integrates signals from O2 to positively regulate cell growth. Hypoxia decreases cell proliferation by repressing mTOR signaling. Insulin-like growth factor I (IGF-I) is a peptide that promotes cell proliferation. A link between hypoxia and IGF-I signaling exists, but a link of IGF-I signaling with mTOR is unclear. Hypoxia induces IGF binding protein-1 (IGFBP-1), a key negative regulator of IGF-I action. Phosphorylation converts IGFBP-1 into a bio-inhibitor of IGF-I action. The mechanisms that regulate IGFBP-1 phosphorylation in hypoxia and inhibit IGF action are largely uncharacterized. PROGRESS AND JUSTIFICATION: Findings from my NSERC-funded research have put-forth a novel concept that hypoxia triggers site-specific IGFBP-1 hyperphosphorylation to inhibit cell proliferation. We have shown that mTOR is an important regulator of IGF-I action. My 2014 NSREC-funded data suggest that hypoxia induced IGFBP-1 phosphorylation may be linked with mTOR. I hypothesize that hypoxia inhibits mTOR, which activates protein kinase CK2 for hypoxia-induced IGFBP-1 phosphorylation and inhibition of IGF-I-action. I will use HepG2 cells and techniques e.g., RNAi/immunoblotting/MRM-MS and functional proteomics to test my hypothesis in 3 Aims. Aim1. Determine that mTORC1 is required in regulating hypoxia-induced IGFBP-1 phosphorylation. We will identify upstream activators of mTORC1 signaling using by siRNA silencing to show the requirement of mTORC1 inhibition in IGFBP-1 phosphorylation and reduction of cell growth via IGF-I action. Aim 2. Determine the role of CK2 in IGFBP-1 phosphorylation. We will use multiple strategies such as co-immunoprecipitation, phospho-site mapping and a CK2 inhibitor resistant mutant to determine that IGFBP-1 is bona fide CK2 substrate. Aim 3. Determine that CK2 is regulated by mTORC1 in hypoxia. We will silence CK2 with/without mTORC1/C2 silencing in normoxia or hypoxia to determine whether mTOR regulates CK2 expression/activity and IGF-I bioactivity in hypoxia. SIGNIFICANCE: This research will provide fundamental knowledge of the interactive cellular mechanisms involving mTOR and CK2 that control IGF action in hypoxia, critical for developmental, physiological, and aberrant processes. HQP: Trainees in my laboratory will receive a strong background in the fundamentals of protein biochemistry, functional-proteomics, and molecular-cell biology to achieve recognition/scholarships and a career in academia/industry/governmental laboratories.