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

Terashima M, Fujita Y, Togashi Y, Sakai K, De Velasco MA, Tomida S, Nishio K - Oncotarget (2014)

Bottom Line: The KIAA1199 gene was first discovered to be associated with non-syndromic hearing loss.A pull-down analysis showed that the glycogen phosphorylase kinase β-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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genome Biology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, Japan.

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 β-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.

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KIAA1199 enhanced the phosphorylation of PYGB in the absence of serum(A) To detect the phosphorylation of PYGB in SNU16, HepG2, and TU-KATOIII cells under serum-free conditions, immunoprecipitation obtained using anti-PYGB antibody was analyzed using western blotting with an antibody against phospho-serine residue. Western blotting analyses for PYGB, KIAA1199, and β-actin were also performed. (B) The phosphorylation of PYGB in TU-KATO III cells was enhanced under serum-free conditions. (C) The interaction of KIAA1199 with PYGB under serum-free conditions was observed in TU-KATO III cells using immunoprecipitation with a PYGB antibody followed by a KIAA1199 antibody.
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Figure 4: KIAA1199 enhanced the phosphorylation of PYGB in the absence of serum(A) To detect the phosphorylation of PYGB in SNU16, HepG2, and TU-KATOIII cells under serum-free conditions, immunoprecipitation obtained using anti-PYGB antibody was analyzed using western blotting with an antibody against phospho-serine residue. Western blotting analyses for PYGB, KIAA1199, and β-actin were also performed. (B) The phosphorylation of PYGB in TU-KATO III cells was enhanced under serum-free conditions. (C) The interaction of KIAA1199 with PYGB under serum-free conditions was observed in TU-KATO III cells using immunoprecipitation with a PYGB antibody followed by a KIAA1199 antibody.

Mentions: Glycogen phosphorylase kinase (PHK) is a serine/threonine-specific protein kinase that phosphorylates a serine residue in glycogen phosphorylase (PYG), triggering the activation of this latter enzyme that catalyzes the phosphorolytic cleavage of the α-1,4 glycosidic linkages of glycogen, releasing glucose-1-phosphate as the reaction product. Since glycogen metabolism has recently been suggested to be a key pathway of metabolic reprogramming in cancer cells [9-12] and PHKB intracellularly binds KIAA1199, as shown in Figure 3, we questioned to what extent KIAA1199 is associated with glycogen breakdown. Several reports have demonstrated that glycogen phosphorylase brain form (PYGB) is overexpressed in various cancer tissues, including gastric cancer [13-15]. Based on this observation, we investigated whether the phosphorylation of endogenous PYGB occurs in SNU16, a cell line expressing a relatively low level of KIAA1199 (Figure 1B). As shown in Fig. 4A, the phosphorylation of PYGB was clearly observed when KIAA1199 was overexpressed in SNU16 (SNU16/KIAA1199), while it was barely detectable in the control cells (SNU16/EGFP). Similar results were obtained for the HepG2 cell line, a hepatocellular cancer cell line that moderately expresses KIAA1199 (Figures 4A and 1B). Interestingly, the phosphorylation of PYGB occurred in intact TU-KATO III cells and was enhanced by serum starvation (Figure 4B). This cell line expresses a relatively high level of KIAA1199, which may account for PYGB's susceptibility to phosphorylation. This hypothesis seems plausible because TU-KATOIII with KIAA1199 silenced by siRNA showed a marked reduction in the phosphorylation of PYGB (Figure 4A). Furthermore, immunoprecipitation showed a moderate interaction of KIAA1199 with PYGB under serum-free conditions (Figure 4C). These results suggest that KIAA1199 interacts with PYGB directly or indirectly through PHKB, resulting in the enhanced phosphorylation of PYGB under serum-free conditions.


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

Terashima M, Fujita Y, Togashi Y, Sakai K, De Velasco MA, Tomida S, Nishio K - Oncotarget (2014)

KIAA1199 enhanced the phosphorylation of PYGB in the absence of serum(A) To detect the phosphorylation of PYGB in SNU16, HepG2, and TU-KATOIII cells under serum-free conditions, immunoprecipitation obtained using anti-PYGB antibody was analyzed using western blotting with an antibody against phospho-serine residue. Western blotting analyses for PYGB, KIAA1199, and β-actin were also performed. (B) The phosphorylation of PYGB in TU-KATO III cells was enhanced under serum-free conditions. (C) The interaction of KIAA1199 with PYGB under serum-free conditions was observed in TU-KATO III cells using immunoprecipitation with a PYGB antibody followed by a KIAA1199 antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: KIAA1199 enhanced the phosphorylation of PYGB in the absence of serum(A) To detect the phosphorylation of PYGB in SNU16, HepG2, and TU-KATOIII cells under serum-free conditions, immunoprecipitation obtained using anti-PYGB antibody was analyzed using western blotting with an antibody against phospho-serine residue. Western blotting analyses for PYGB, KIAA1199, and β-actin were also performed. (B) The phosphorylation of PYGB in TU-KATO III cells was enhanced under serum-free conditions. (C) The interaction of KIAA1199 with PYGB under serum-free conditions was observed in TU-KATO III cells using immunoprecipitation with a PYGB antibody followed by a KIAA1199 antibody.
Mentions: Glycogen phosphorylase kinase (PHK) is a serine/threonine-specific protein kinase that phosphorylates a serine residue in glycogen phosphorylase (PYG), triggering the activation of this latter enzyme that catalyzes the phosphorolytic cleavage of the α-1,4 glycosidic linkages of glycogen, releasing glucose-1-phosphate as the reaction product. Since glycogen metabolism has recently been suggested to be a key pathway of metabolic reprogramming in cancer cells [9-12] and PHKB intracellularly binds KIAA1199, as shown in Figure 3, we questioned to what extent KIAA1199 is associated with glycogen breakdown. Several reports have demonstrated that glycogen phosphorylase brain form (PYGB) is overexpressed in various cancer tissues, including gastric cancer [13-15]. Based on this observation, we investigated whether the phosphorylation of endogenous PYGB occurs in SNU16, a cell line expressing a relatively low level of KIAA1199 (Figure 1B). As shown in Fig. 4A, the phosphorylation of PYGB was clearly observed when KIAA1199 was overexpressed in SNU16 (SNU16/KIAA1199), while it was barely detectable in the control cells (SNU16/EGFP). Similar results were obtained for the HepG2 cell line, a hepatocellular cancer cell line that moderately expresses KIAA1199 (Figures 4A and 1B). Interestingly, the phosphorylation of PYGB occurred in intact TU-KATO III cells and was enhanced by serum starvation (Figure 4B). This cell line expresses a relatively high level of KIAA1199, which may account for PYGB's susceptibility to phosphorylation. This hypothesis seems plausible because TU-KATOIII with KIAA1199 silenced by siRNA showed a marked reduction in the phosphorylation of PYGB (Figure 4A). Furthermore, immunoprecipitation showed a moderate interaction of KIAA1199 with PYGB under serum-free conditions (Figure 4C). These results suggest that KIAA1199 interacts with PYGB directly or indirectly through PHKB, resulting in the enhanced phosphorylation of PYGB under serum-free conditions.

Bottom Line: The KIAA1199 gene was first discovered to be associated with non-syndromic hearing loss.A pull-down analysis showed that the glycogen phosphorylase kinase β-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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genome Biology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, Japan.

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 β-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.

Show MeSH
Related in: MedlinePlus