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

摘要

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-2 beta, 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-2 beta and a nuclear co-localization between CSNK-2 beta and mTOR. Proximity ligation assay (PLA) indicated proximity between IGFBP-1 and CSNK-2 beta as well as mTOR and CSNK-2 beta but not between mTOR and IGFBP-1. Three-dimensional rendering of the PLA images validated that IGFBP-1 and CSNK-2 beta interactions were in the perinuclear region and mTOR and CSNK-2 beta interactions were also predominantly perinuclear rather than nuclear as indicated by mTOR and CSNK-2 beta co-localization. Compared with control, hypoxia and rapamycin treatment showed markedly amplified PLA signals for IGFBP-1 and CSNK-2 beta (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-2 beta and IGFBP-1 as well as mTOR and CSNK-2 beta, providing strong evidence of a mechanistic link between mTOR and IGF-I signaling, two critical regulators of cell growth via CSNK-2.