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Identification of C-terminal Hsp70-interacting protein as a mediator of tumour necrosis factor action in osteoblast differentiation by targeting osterix for degradation.

Xie J, Gu J - J. Cell. Mol. Med. (2015)

Bottom Line: C2C12, MC3T3-E1 and HEK293T cell lines were cultured and cotransfected with related plasmids.The expression levels of TNF-α-induced CHIP and Osx were examined by RT-PCR and Western blot analysis.Co-immunoprecipitation and ubiquitination assays revealed ubiquitinated Osx, confirmed that CHIP indeed interacted with Osx and identified K55 and K386 residues as the ubiquitination sites in Osx, Luciferase reporter assay and Small interfering RNAs examined whether TNF-α target the bone morphogenetic protein signalling through CHIP.

View Article: PubMed Central - PubMed

Affiliation: Department of Rheumatology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, China.

No MeSH data available.


Related in: MedlinePlus

The residues K55 and K386 are the ubiquitination sites of Osx. (A) Mouse Osx primary sequence with the lysine residues indicated. (B) HEK 293T cells were cotransfected with HA-Ub expression plasmids and the WT Flag-Osx plasmid or the K26R, K55R, K111R, K227R, K229R, K244R, K386R or K414R mutants. Cell lysates were immunoprecipitated using an anti-Flag antibody and blotted with an anti-HA antibody. The results are shown for one of three independent experiments. (C) HEK 293T cells were cotransfected with the Ocn-Luc reporter, control Renilla luciferase reporter vector, and the WT Flag-Osx plasmid or Lys-to-Arg mutants. The relative luciferase activity was measured 24 hrs after transfection. The results were obtained from three independent experiments performed in triplicate. The data are expressed as the mean ± SD.
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fig04: The residues K55 and K386 are the ubiquitination sites of Osx. (A) Mouse Osx primary sequence with the lysine residues indicated. (B) HEK 293T cells were cotransfected with HA-Ub expression plasmids and the WT Flag-Osx plasmid or the K26R, K55R, K111R, K227R, K229R, K244R, K386R or K414R mutants. Cell lysates were immunoprecipitated using an anti-Flag antibody and blotted with an anti-HA antibody. The results are shown for one of three independent experiments. (C) HEK 293T cells were cotransfected with the Ocn-Luc reporter, control Renilla luciferase reporter vector, and the WT Flag-Osx plasmid or Lys-to-Arg mutants. The relative luciferase activity was measured 24 hrs after transfection. The results were obtained from three independent experiments performed in triplicate. The data are expressed as the mean ± SD.

Mentions: Because the mutation of Lys residues may induce structural changes in the Osx protein and also affect its activity by blocking ubiquitination, each of the Lys residues of Osx was replaced with an Arg residue (Fig.4A), which maintained the positive charge but could not serve as an acceptor site for ubiquitination. To identify the ubiquitination sites in Osx, Osx-deficient HEK293T cells were cotransfected with the WT Flag-Osx plasmid, mutants with different Lys-to-Arg mutations and Ocn-Luc constructs. Overexpression of the K26R, K55R, K111R, K227R, K229R, K244R, K386R and K414R mutants increased Ocn-dependent luciferase reporter gene expression by more than 2.6-fold, compared with WT Osx. Eight of 22 Lys residues in Osx were able to accept the ubiquitin molecule (Fig.4C), and the Lys-to-Arg mutations at these sites might abrogate Osx ubiquitination and degradation. To further analyse the Ub Lys site(s), we cotransfected the WT Flag-Osx or the eight above-described Lys-to-Arg mutants, along with a HA-ubiquitin vector, into HEK293T cells. We found that only the K55R and K386R mutants displayed dramatically decreased ubiquitiation. We demonstrated that K55 and K386 are the ubiquitination sites of Osx by co-immunoprecipitation assays. These results indicated that the Osx K55R and K386R mutations reduced Osx ubiquitination activity to a level comparable with the result obtained with the wild-type control (Fig.4B), suggesting that these two residues may be ubiquitination sites of Osx.


Identification of C-terminal Hsp70-interacting protein as a mediator of tumour necrosis factor action in osteoblast differentiation by targeting osterix for degradation.

Xie J, Gu J - J. Cell. Mol. Med. (2015)

The residues K55 and K386 are the ubiquitination sites of Osx. (A) Mouse Osx primary sequence with the lysine residues indicated. (B) HEK 293T cells were cotransfected with HA-Ub expression plasmids and the WT Flag-Osx plasmid or the K26R, K55R, K111R, K227R, K229R, K244R, K386R or K414R mutants. Cell lysates were immunoprecipitated using an anti-Flag antibody and blotted with an anti-HA antibody. The results are shown for one of three independent experiments. (C) HEK 293T cells were cotransfected with the Ocn-Luc reporter, control Renilla luciferase reporter vector, and the WT Flag-Osx plasmid or Lys-to-Arg mutants. The relative luciferase activity was measured 24 hrs after transfection. The results were obtained from three independent experiments performed in triplicate. The data are expressed as the mean ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4549032&req=5

fig04: The residues K55 and K386 are the ubiquitination sites of Osx. (A) Mouse Osx primary sequence with the lysine residues indicated. (B) HEK 293T cells were cotransfected with HA-Ub expression plasmids and the WT Flag-Osx plasmid or the K26R, K55R, K111R, K227R, K229R, K244R, K386R or K414R mutants. Cell lysates were immunoprecipitated using an anti-Flag antibody and blotted with an anti-HA antibody. The results are shown for one of three independent experiments. (C) HEK 293T cells were cotransfected with the Ocn-Luc reporter, control Renilla luciferase reporter vector, and the WT Flag-Osx plasmid or Lys-to-Arg mutants. The relative luciferase activity was measured 24 hrs after transfection. The results were obtained from three independent experiments performed in triplicate. The data are expressed as the mean ± SD.
Mentions: Because the mutation of Lys residues may induce structural changes in the Osx protein and also affect its activity by blocking ubiquitination, each of the Lys residues of Osx was replaced with an Arg residue (Fig.4A), which maintained the positive charge but could not serve as an acceptor site for ubiquitination. To identify the ubiquitination sites in Osx, Osx-deficient HEK293T cells were cotransfected with the WT Flag-Osx plasmid, mutants with different Lys-to-Arg mutations and Ocn-Luc constructs. Overexpression of the K26R, K55R, K111R, K227R, K229R, K244R, K386R and K414R mutants increased Ocn-dependent luciferase reporter gene expression by more than 2.6-fold, compared with WT Osx. Eight of 22 Lys residues in Osx were able to accept the ubiquitin molecule (Fig.4C), and the Lys-to-Arg mutations at these sites might abrogate Osx ubiquitination and degradation. To further analyse the Ub Lys site(s), we cotransfected the WT Flag-Osx or the eight above-described Lys-to-Arg mutants, along with a HA-ubiquitin vector, into HEK293T cells. We found that only the K55R and K386R mutants displayed dramatically decreased ubiquitiation. We demonstrated that K55 and K386 are the ubiquitination sites of Osx by co-immunoprecipitation assays. These results indicated that the Osx K55R and K386R mutations reduced Osx ubiquitination activity to a level comparable with the result obtained with the wild-type control (Fig.4B), suggesting that these two residues may be ubiquitination sites of Osx.

Bottom Line: C2C12, MC3T3-E1 and HEK293T cell lines were cultured and cotransfected with related plasmids.The expression levels of TNF-α-induced CHIP and Osx were examined by RT-PCR and Western blot analysis.Co-immunoprecipitation and ubiquitination assays revealed ubiquitinated Osx, confirmed that CHIP indeed interacted with Osx and identified K55 and K386 residues as the ubiquitination sites in Osx, Luciferase reporter assay and Small interfering RNAs examined whether TNF-α target the bone morphogenetic protein signalling through CHIP.

View Article: PubMed Central - PubMed

Affiliation: Department of Rheumatology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, China.

No MeSH data available.


Related in: MedlinePlus