PMID- 29037858 OWN - NLM STAT- MEDLINE DCOM- 20180109 LR - 20240213 IS - 1525-2191 (Electronic) IS - 0002-9440 (Print) IS - 0002-9440 (Linking) VI - 188 IP - 1 DP - 2018 Jan TI - Co-Localization of Insulin-Like Growth Factor Binding Protein-1, Casein Kinase-2beta, and Mechanistic Target of Rapamycin in Human Hepatocellular Carcinoma Cells as Demonstrated by Dual Immunofluorescence and in Situ Proximity Ligation Assay. PG - 111-124 LID - S0002-9440(17)30532-1 [pii] LID - 10.1016/j.ajpath.2017.09.009 [doi] AB - 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-2beta, 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-2beta and a nuclear co-localization between CSNK-2beta and mTOR. Proximity ligation assay (PLA) indicated proximity between IGFBP-1 and CSNK-2beta as well as mTOR and CSNK-2beta but not between mTOR and IGFBP-1. Three-dimensional rendering of the PLA images validated that IGFBP-1 and CSNK-2beta interactions were in the perinuclear region and mTOR and CSNK-2beta interactions were also predominantly perinuclear rather than nuclear as indicated by mTOR and CSNK-2beta co-localization. Compared with control, hypoxia and rapamycin treatment showed markedly amplified PLA signals for IGFBP-1 and CSNK-2beta (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-2beta and IGFBP-1 as well as mTOR and CSNK-2beta, providing strong evidence of a mechanistic link between mTOR and IGF-I signaling, two critical regulators of cell growth via CSNK-2. CI - Copyright (c) 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. FAU - Singal, Sahil S AU - Singal SS AD - Department of Biochemistry, University of Western Ontario, London, Ontario, Canada. FAU - Nygard, Karen AU - Nygard K AD - Biotron Laboratory, University of Western Ontario, London, Ontario, Canada. FAU - Dhruv, Manthan R AU - Dhruv MR AD - Department of Pediatrics, University of Western Ontario, London, Ontario, Canada. FAU - Biggar, Kyle AU - Biggar K AD - Department of Biochemistry, University of Western Ontario, London, Ontario, Canada; Institute of Biochemistry, Carleton University, Ottawa, Ontario, Canada. FAU - Shehab, Majida A AU - Shehab MA AD - Department of Pediatrics, University of Western Ontario, London, Ontario, Canada. FAU - Li, Shawn S-C AU - Li SS AD - Department of Biochemistry, University of Western Ontario, London, Ontario, Canada. FAU - Jansson, Thomas AU - Jansson T AD - Department of Obstetrics & Gynecology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, Canada. FAU - Gupta, Madhulika B AU - Gupta MB AD - 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. LA - eng GR - R03 HD078313/HD/NICHD NIH HHS/United States PT - Journal Article DEP - 20171014 PL - United States TA - Am J Pathol JT - The American journal of pathology JID - 0370502 RN - 0 (Insulin-Like Growth Factor Binding Protein 1) RN - EC 2.7.1.1 (MTOR protein, human) RN - EC 2.7.11.1 (Casein Kinase II) RN - EC 2.7.11.1 (TOR Serine-Threonine Kinases) SB - IM MH - Carcinoma, Hepatocellular/*metabolism/pathology MH - Casein Kinase II/*metabolism MH - Fluorescent Antibody Technique MH - Hep G2 Cells MH - Humans MH - Insulin-Like Growth Factor Binding Protein 1/*metabolism MH - Liver Neoplasms/*metabolism/pathology MH - Phosphorylation MH - Signal Transduction/physiology MH - TOR Serine-Threonine Kinases/*metabolism PMC - PMC5745526 EDAT- 2017/10/19 06:00 MHDA- 2018/01/10 06:00 PMCR- 2019/01/01 CRDT- 2017/10/18 06:00 PHST- 2017/05/19 00:00 [received] PHST- 2017/08/05 00:00 [revised] PHST- 2017/09/07 00:00 [accepted] PHST- 2017/10/19 06:00 [pubmed] PHST- 2018/01/10 06:00 [medline] PHST- 2017/10/18 06:00 [entrez] PHST- 2019/01/01 00:00 [pmc-release] AID - S0002-9440(17)30532-1 [pii] AID - 10.1016/j.ajpath.2017.09.009 [doi] PST - ppublish SO - Am J Pathol. 2018 Jan;188(1):111-124. doi: 10.1016/j.ajpath.2017.09.009. Epub 2017 Oct 14.