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

• Published in: The American journal of pathology Vol. 188; no. 1; pp. 111 - 124
• Main Authors: Singal, Sahil S., Nygard, Karen, Dhruv, Manthan R., Biggar, Kyle, Shehab, Majida A., Li, Shawn S.-C., Jansson, Thomas, Gupta, Madhulika B.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2018; American Society for Investigative Pathology
• Subjects: Carcinoma, Hepatocellular - metabolism; Carcinoma, Hepatocellular - pathology; Casein Kinase II - metabolism; Fluorescent Antibody Technique; Hep G2 Cells; Humans; Insulin-Like Growth Factor Binding Protein 1 - metabolism; Liver Neoplasms - metabolism; Liver Neoplasms - pathology; Phosphorylation; Signal Transduction - physiology; TOR Serine-Threonine Kinases - metabolism
• ISSN: 0002-9440; 1525-2191
• DOI: 10.1016/j.ajpath.2017.09.009

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.