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

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.

Novel roles of mechanistic target of rapamycin signaling in regulating fetal growth†

“…Moreover, how the phosphorylation of IGFBP-1 is regulated has, until recently, remained unknown. Based on studies in cultured HepG2 cells and primary fetal baboon hepatocytes, we demonstrated that inhibition of mTOR is required for increased IGFBP-1 secretion and phosphorylation in response to hypoxia [83,84] and the enhanced IGFBP-1 secretion following decreased amino acid availability [85]. In contrast, IGFBP-1 hyperphosphorylation in response to amino acid deprivation is mediated by activation of the AAR signaling pathway [85] (Figure 1).…”

“…Moreover, it was demonstrated that CK2 and PKC constitute the key kinases, regulated by mTOR and AAR, responsible for IGFBP-1 serine phosphorylation [52,84,87] (Figure 1). An important role of CK2 in the phosphorylation of IGFBP-1 was further supported by extensive co-localization between these two proteins in HepG2 cells ( Figure 2).…”

Increased talin–vinculin spatial proximities in livers in response to spotted fever group rickettsial and Ebola virus infections

“…When actin filaments are pulled, the R3 domain will undergo a conformational change, which causes RIAM to dissociate and vinculin to bind [7,41]. Talin is a mechanosensitive molecule and this mechanosensitivity relates to the interaction of talin with vinculin [42,43]. Vinculin is a major talin-binding partner that facilitates crosstalk between talin and actin through its talin-binding head domain (V-head) and its actin-binding tail domain (V-tail) [44,45].…”

Dairy consumption and hepatocellular carcinoma risk

“…The nutrient-sensitive kinase mechanistic target of rapamycin complex 1 (mTORC1) is overactivated in many cancers including HCC (70)(71)(72)(73)(74)(75)(76)(77)(78)(79). Not only insulin and IGF-1 activate mTORC1 but also AAs (80,81), preferentially the BCAAs leucine, isoleucine and valine (82)(83)(84)(85)(86)(87)(88).…”