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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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Construction of MBP-KIAA1199 fusion protein and detection KIAA1199 binding protein using a pull down assay(A) The schematic structure of five fragments of KIAA1199 used to fuse with MBP. The numbers represent the positions of the amino acids. All these fragments consisted of approximately 300 amino acids. (B) A pull down assay was performed using the fusion proteins with TU-KATO III whole cell lysate. The candidates were separated using SDS-PAGE, visualized using silver staining, and then analyzed using mass spectrometry. (C) An immunoprecipitation analysis was performed to confirm the interaction of COPA and PHKB with KIAA1199 in TU-KATO III cells.
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Figure 3: Construction of MBP-KIAA1199 fusion protein and detection KIAA1199 binding protein using a pull down assay(A) The schematic structure of five fragments of KIAA1199 used to fuse with MBP. The numbers represent the positions of the amino acids. All these fragments consisted of approximately 300 amino acids. (B) A pull down assay was performed using the fusion proteins with TU-KATO III whole cell lysate. The candidates were separated using SDS-PAGE, visualized using silver staining, and then analyzed using mass spectrometry. (C) An immunoprecipitation analysis was performed to confirm the interaction of COPA and PHKB with KIAA1199 in TU-KATO III cells.

Mentions: To elucidate the biological function of KIAA1199, we constructed five variants of MBP-KIAA1199 fusion protein, as shown in Figure 3A, and forcibly expressed these variants in E. coli. Pull down assays were performed using purified recombinant proteins or MBP alone (mock) using a whole cell lysate of TU-KATOIII, a cell line expressing a high level of endogenous KIAA1199. The pull-down fraction of MBP-5, which corresponds to the C-terminal region of KIAA1199 protein, revealed two distinct protein bands, compared with the mock pull-down (Figure 3B). To identify the amino acid sequences of these putative KIAA1199 interacting proteins, the candidate bands were cut out from the gel and digested, and the resulting peptide fragments were analyzed using mass spectrometry. Peptide mass finger printing and tandem mass spectrum data from MALDI-TOF/MS were analyzed by searching against an MSDB or NCBI-nr database using MASCOT (Matrix Science, London, UK) search software. As described in Fig. 3B, these interacting proteins were identified as coatomer protein complex α-subunit (COPA, 140 kDa) and glycogen phosphorylase kinase β-subunit (PHKB, 125 kDa). The interactions of these proteins with KIAA1199 were confirmed using immunoprecipitation with the respective antibodies (Figure 3C).


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)

Construction of MBP-KIAA1199 fusion protein and detection KIAA1199 binding protein using a pull down assay(A) The schematic structure of five fragments of KIAA1199 used to fuse with MBP. The numbers represent the positions of the amino acids. All these fragments consisted of approximately 300 amino acids. (B) A pull down assay was performed using the fusion proteins with TU-KATO III whole cell lysate. The candidates were separated using SDS-PAGE, visualized using silver staining, and then analyzed using mass spectrometry. (C) An immunoprecipitation analysis was performed to confirm the interaction of COPA and PHKB with KIAA1199 in TU-KATO III cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196182&req=5

Figure 3: Construction of MBP-KIAA1199 fusion protein and detection KIAA1199 binding protein using a pull down assay(A) The schematic structure of five fragments of KIAA1199 used to fuse with MBP. The numbers represent the positions of the amino acids. All these fragments consisted of approximately 300 amino acids. (B) A pull down assay was performed using the fusion proteins with TU-KATO III whole cell lysate. The candidates were separated using SDS-PAGE, visualized using silver staining, and then analyzed using mass spectrometry. (C) An immunoprecipitation analysis was performed to confirm the interaction of COPA and PHKB with KIAA1199 in TU-KATO III cells.
Mentions: To elucidate the biological function of KIAA1199, we constructed five variants of MBP-KIAA1199 fusion protein, as shown in Figure 3A, and forcibly expressed these variants in E. coli. Pull down assays were performed using purified recombinant proteins or MBP alone (mock) using a whole cell lysate of TU-KATOIII, a cell line expressing a high level of endogenous KIAA1199. The pull-down fraction of MBP-5, which corresponds to the C-terminal region of KIAA1199 protein, revealed two distinct protein bands, compared with the mock pull-down (Figure 3B). To identify the amino acid sequences of these putative KIAA1199 interacting proteins, the candidate bands were cut out from the gel and digested, and the resulting peptide fragments were analyzed using mass spectrometry. Peptide mass finger printing and tandem mass spectrum data from MALDI-TOF/MS were analyzed by searching against an MSDB or NCBI-nr database using MASCOT (Matrix Science, London, UK) search software. As described in Fig. 3B, these interacting proteins were identified as coatomer protein complex α-subunit (COPA, 140 kDa) and glycogen phosphorylase kinase β-subunit (PHKB, 125 kDa). The interactions of these proteins with KIAA1199 were confirmed using immunoprecipitation with the respective antibodies (Figure 3C).

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