4 years ago

Co-Localization of IGFBP-1, Protein Kinase CSNK-2β, and mTOR in HepG2 Cells as Demonstrated by Dual Immunofluorescence and in Situ Proximity Ligation Assay

Insulin-like growth factor binding protein (IGFBP)-1 influences fetal growth by modifying IGF-I bioavailability. IGFBP-1 phosphorylation, which markedly increases its affinity for IGF-I, is regulated by mTOR and CSNK-2. However, the underlying molecular mechanisms remain unknown. We examined the cellular localization and potential interactions of IGFBP-1, CSNK-2β, and mTOR as a prerequisite for protein-protein interaction. Confocal/Z-stack analysis of dual immunofluorescence images indicated a potential perinuclear co-localization between IGFBP-1+CSNK-2β whereas a nuclear co-localization between CSNK-2β+mTOR. Co-immunoprecipitation validated these associations. Proximity ligation assay (PLA) corroborated immunohistochemistry data indicating proximity (<40 nm) between IGFBP-1+CSNK-2β and mTOR+CSNK-2β, but not between mTOR+IGFBP-1. Three-dimensional rendering of the PLA images validated that interactions of IGFBP-1+CSNK-2β were in the perinuclear region. Three-dimensional cutaways PLA images showed that mTOR+CSNK-2β interactions were also predominantly perinuclear rather than nuclear as indicated by mTOR+CSNK-2β co-localization. Compared to the control, combined hypoxia+rapamycin treatment showed markedly amplified PLA signals for IGFBP-1+CSNK-2β (approximately 18-fold, P = 0.0002). Quantitative stable isotope labeling with multiple reaction monitoring-mass spectrometry demonstrated that hypoxia+rapamycin increased IGFBP-1 phosphorylation at Ser98/101/119 and at a novel site Ser174, but most dramatically (106-fold) at Ser169. Using multiple complementary approaches, we report data showing interactions between CSNK-2β and IGFBP-1 as well as mTOR and CSNK-2β, respectively, providing strong supporting evidence for a novel mechanistic link between mTOR and IGF-I signaling, two critical regulators of cell growth via CSNK-2.

Publisher URL: www.sciencedirect.com/science

DOI: S0002944017305321

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