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Am J Pathol. 2018 Jan;188(1):111-124. doi: 10.1016/j.ajpath.2017.09.009. Epub 2017 Oct 14.

Co-Localization of Insulin-Like Growth Factor Binding Protein-1, Casein Kinase-2β, and Mechanistic Target of Rapamycin in Human Hepatocellular Carcinoma Cells as Demonstrated by Dual Immunofluorescence and in Situ Proximity Ligation Assay.

Author information

1
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada.
2
Biotron Laboratory, University of Western Ontario, London, Ontario, Canada.
3
Department of Pediatrics, University of Western Ontario, London, Ontario, Canada.
4
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada; Institute of Biochemistry, Carleton University, Ottawa, Ontario, Canada.
5
Department of Obstetrics & Gynecology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, Canada.
6
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada; Department of Pediatrics, University of Western Ontario, London, Ontario, Canada; Children's Health Research Institute, London, Ontario, Canada. Electronic address: mbgupta@uwo.ca.

Abstract

Insulin-like growth factor binding protein (IGFBP)-1 influences fetal growth by modifying insulin-like growth factor-I (IGF-I) bioavailability. IGFBP-1 phosphorylation, which markedly increases its affinity for IGF-I, is regulated by mechanistic target of rapamycin (mTOR) and casein kinase (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. Analysis of dual immunofluorescence images indicated a potential perinuclear co-localization between IGFBP-1 and CSNK-2β and a nuclear co-localization between CSNK-2β and mTOR. Proximity ligation assay (PLA) indicated proximity between IGFBP-1 and CSNK-2β as well as mTOR and CSNK-2β but not between mTOR and IGFBP-1. Three-dimensional rendering of the PLA images validated that IGFBP-1 and CSNK-2β interactions were in the perinuclear region and mTOR and CSNK-2β interactions were also predominantly perinuclear rather than nuclear as indicated by mTOR and CSNK-2β co-localization. Compared with control, hypoxia and rapamycin treatment showed markedly amplified PLA signals for IGFBP-1 and CSNK-2β (approximately 18-fold, P = 0.0002). Stable isotope labeling with multiple reaction monitoring-mass spectrometry demonstrated that hypoxia and rapamycin treatment increased IGFBP-1 phosphorylation at Ser98/Ser101/Ser119/Ser174 but most considerably (106-fold) at Ser169. We report interactions between CSNK-2β and IGFBP-1 as well as mTOR and CSNK-2β, providing strong evidence of a mechanistic link between mTOR and IGF-I signaling, two critical regulators of cell growth via CSNK-2.

PMID:
29037858
PMCID:
PMC5745526
DOI:
10.1016/j.ajpath.2017.09.009
[Indexed for MEDLINE]
Free PMC Article

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