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KIAA1199 interacts with glycogen phosphorylase kinase ?-subunit (PHKB) to promote glycogen breakdown and cancer cell survival.

ABSTRACT: The KIAA1199 gene was first discovered to be associated with non-syndromic hearing loss. Recently, several reports have shown that the up-regulation of KIAA1199 is associated with cancer cell migration or invasion and a poor prognosis. These findings indicate that KIAA1199 may be a novel target for cancer therapy. Therefore, we explored in detail the function of KIAA1199 in cancer cells. In this study, we investigated the interaction of KIAA1199 protein with intracellular proteins in cancer cells. To this end, we expressed KIAA1199-MBP fusion protein and performed a pull-down assay. In addition, KIAA1199-overexpressing cancer cell lines were constructed using a retroviral vector and were used for further experiments. A pull-down analysis showed that the glycogen phosphorylase kinase ?-subunit (PHKB) interacted with the C-terminal region of KIAA1199 protein. Furthermore, we observed the interaction of KIAA1199 with glycogen phosphorylase brain form (PYGB) under serum-free conditions. The interaction promoted glycogen breakdown and cancer cell survival. Our findings indicate that KIAA1199 plays an important role in glycogen breakdown and cancer cell survival and that it may represent a novel target for cancer therapy.

SUBMITTER: Terashima M

PROVIDER: S-EPMC4196182 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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KIAA1199 interacts with glycogen phosphorylase kinase β-subunit (PHKB) to promote glycogen breakdown and cancer cell survival.

Terashima Masato M Fujita Yoshihiko Y Togashi Yosuke Y Sakai Kazuko K De Velasco Marco A MA Tomida Shuta S Nishio Kazuto K

Oncotarget 20140801 16

The KIAA1199 gene was first discovered to be associated with non-syndromic hearing loss. Recently, several reports have shown that the up-regulation of KIAA1199 is associated with cancer cell migration or invasion and a poor prognosis. These findings indicate that KIAA1199 may be a novel target for cancer therapy. Therefore, we explored in detail the function of KIAA1199 in cancer cells. In this study, we investigated the interaction of KIAA1199 protein with intracellular proteins in cancer cell ...[more]

PMID: 25051373

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Project description:Skeletal muscle phosphorylase kinase (PhK) is an (alphabetagammadelta) 4 hetero-oligomeric enzyme complex that phosphorylates and activates glycogen phosphorylase b (GP b) in a Ca (2+)-dependent reaction that couples muscle contraction with glycogen breakdown. GP b is PhK's only known in vivo substrate; however, given the great size and multiple subunits of the PhK complex, we screened muscle extracts for other potential targets. Extracts of P/J (control) and I/lnJ (PhK deficient) mice were incubated with [gamma- (32)P]ATP with or without Ca (2+) and compared to identify potential substrates. Candidate targets were resolved by two-dimensional polyacrylamide gel electrophoresis, and phosphorylated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified by matrix-assisted laser desorption ionization mass spectroscopy. In vitro studies showed GAPDH to be a Ca (2+)-dependent substrate of PhK, although the rate of phosphorylation is very slow. GAPDH does, however, bind tightly to PhK, inhibiting at low concentrations (IC 50 approximately 0.45 microM) PhK's conversion of GP b. When a short synthetic peptide substrate was substituted for GP b, the inhibition was negligible, suggesting that GAPDH may inhibit predominantly by binding to the PhK complex at a locus distinct from its active site on the gamma subunit. To test this notion, the PhK-GAPDH complex was incubated with a chemical cross-linker, and a dimer between the regulatory beta subunit of PhK and GAPDH was formed. This interaction was confirmed by the fact that a subcomplex of PhK missing the beta subunit, specifically an alphagammadelta subcomplex, was unable to phosphorylate GAPDH, even though it is catalytically active toward GP b. Moreover, GAPDH had no effect on the conversion of GP b by the alphagammadelta subcomplex. The interactions described herein between the beta subunit of PhK and GAPDH provide a possible mechanism for the direct linkage of glycogenolysis and glycolysis in skeletal muscle.

Project description:Background and purposeGlycogen phosphorylase (GP) is the key enzyme for glycogen degradation. GP inhibitors (GPi-s) are glucose lowering agents that cause the accumulation of glucose in the liver as glycogen. Glycogen metabolism has implications in beta cell function. Glycogen degradation can maintain cellular glucose levels, which feeds into catabolism to maintain insulin secretion, and elevated glycogen degradation levels contribute to glucotoxicity. The purpose of this study was to assess whether influencing glycogen metabolism in beta cells by GPi-s affects the function of these cells.Experimental approachThe effects of structurally different GPi-s were investigated on MIN6 insulinoma cells and in a mouse model of diabetes.Key resultsGPi treatment increased glycogen content and, consequently, the surface area of glycogen in MIN6 cells. Furthermore, GPi treatment induced insulin receptor β (InsRβ), Akt and p70S6K phosphorylation, as well as pancreatic and duodenal homeobox 1(PDX1) and insulin expression. In line with these findings, GPi-s enhanced non-stimulated and glucose-stimulated insulin secretion in MIN6 cells. The InsRβ was shown to co-localize with glycogen particles as confirmed by in silico screening, where components of InsR signalling were identified as glycogen-bound proteins. GPi-s also activated the pathway of insulin secretion, indicated by enhanced glycolysis, mitochondrial oxidation and calcium signalling. Finally, GPi-s increased the size of islets of Langerhans and improved glucose-induced insulin release in mice.Conclusion and implicationsThese data suggest that GPi-s also target beta cells and can be repurposed as agents to preserve beta cell function or even ameliorate beta cell dysfunction in different forms of diabetes.Linked articlesThis article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

Dataset Information

KIAA1199 interacts with glycogen phosphorylase kinase ?-subunit (PHKB) to promote glycogen breakdown and cancer cell survival.

Publications

KIAA1199 interacts with glycogen phosphorylase kinase β-subunit (PHKB) to promote glycogen breakdown and cancer cell survival.

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