%0 Journal Article
%T Co-Localization of Insulin-Like Growth Factor Binding Protein-1, Casein Kinase-2¦Â, and Mechanistic Target of Rapamycin in Human Hepatocellular Carcinoma Cells as Demonstrated by Dual Immunofluorescence and in Situ Proximity Ligation Assay
%A Karen Nygard
%A Kyle Biggar
%A Madhulika B. Gupta
%A Majida A. Shehab
%A Manthan R. Dhruv
%A Sahil S. Singal
%A Shawn S.-C. Li
%A Thomas Jansson
%J Archive of "The American Journal of Pathology".
%D 2018
%R 10.1016/j.ajpath.2017.09.009
%X Insulin-like growth factor binding protein (IGFBP)¨C1 influences fetal growth by modifying insulin-like growth factor¨CI (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)¨C2. However, the underlying molecular mechanisms remain unknown. We examined the cellular localization and potential interactions of IGFBP-1, CSNK-2¦Â, and mTOR as a prerequisite for protein¨Cprotein interaction. Analysis of dual immunofluorescence images indicated a potential perinuclear co-localization between IGFBP-1 and CSNK-2¦Â and a nuclear co-localization between CSNK-2¦Â and mTOR. Proximity ligation assay (PLA) indicated proximity between IGFBP-1 and CSNK-2¦Â as well as mTOR and CSNK-2¦Â but not between mTOR and IGFBP-1. Three-dimensional rendering of the PLA images validated that IGFBP-1 and CSNK-2¦Â interactions were in the perinuclear region and mTOR and CSNK-2¦Â interactions were also predominantly perinuclear rather than nuclear as indicated by mTOR and CSNK-2¦Â co-localization. Compared with control, hypoxia and rapamycin treatment showed markedly amplified PLA signals for IGFBP-1 and CSNK-2¦Â (approximately 18-fold, P = 0.0002). Stable isotope labeling with multiple reaction monitoring¨Cmass 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-2¦Â and IGFBP-1 as well as mTOR and CSNK-2¦Â, providing strong evidence of a mechanistic link between mTOR and IGF-I signaling, two critical regulators of cell growth via CSNK-2
%U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5745526/