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Lysine Methylation of the Valosin-Containing Protein (VCP) Is Dispensable for Development and Survival of Mice.

Fusser M, Kernstock S, Aileni VK, Egge-Jacobsen W, Falnes PØ, Klungland A - PLoS ONE (2015)

Bottom Line: We show that Vcpkmt is ubiquitously expressed in all tissues examined and confirm the sub-cellular localization to the cytoplasm.The results show that VCPKMT is a highly specific enzyme, and suggest that VCP is its sole substrate.Overall the results show that VCPKMT is an enzyme required for methylation of K315 of VCP in vivo, but VCPKMT is not essential for development or survival under unstressed conditions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Oslo University Hospital, Rikshospitalet, Oslo, Norway.

ABSTRACT
Valosin-containing protein (VCP) is a homohexameric ATPase involved in a multitude cellular processes and it was recently shown that VCP is trimethylated at lysine 315 by the VCP lysine methyltransferase (VCPKMT). Here, we generated and validated a constitutive knockout mouse by targeting exon 1-4 of the Vcpkmt gene. We show that Vcpkmt is ubiquitously expressed in all tissues examined and confirm the sub-cellular localization to the cytoplasm. We show by (I) mass spectrometric analysis, (II) VCPKMT-mediated in vitro methylation of VCP in cell extracts and (III) immunostaining with a methylation specific antibody, that in Vcpkmt-/- mice the methylation of lysine 315 in VCP is completely abolished. In contrast, VCP is almost exclusively trimethylated in wild-type mice. Furthermore, we investigated the specificity of VCPKMT with in vitro methylation assays using as source of substrate protein extracts from Vcpkmt-/- mouse organs or three human Vcpkmt-/- cell lines. The results show that VCPKMT is a highly specific enzyme, and suggest that VCP is its sole substrate. The Vcpkmt-/- mice were viable, fertile and had no obvious pathological phenotype. Their body weight, life span and acute endurance capacity were comparable to wild-type controls. Overall the results show that VCPKMT is an enzyme required for methylation of K315 of VCP in vivo, but VCPKMT is not essential for development or survival under unstressed conditions.

No MeSH data available.


Related in: MedlinePlus

Immunostaining of K315me3-VCP in cells and tissues of wild-type and Vcpkmt-/- mice.Immunostaining of K315me3-VCP (green), total VCP (red) and DAPI DNA counterstain (blue) of wild-type and Vcpkmt-/-. Scale bars 20 μm. (A) Cultured mouse embryonic fibroblast (MEFs). (B) Semniferous tubuli of testes. (C) Hepatocytes with a central vein. (D) Kidney cortex.
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pone.0141472.g003: Immunostaining of K315me3-VCP in cells and tissues of wild-type and Vcpkmt-/- mice.Immunostaining of K315me3-VCP (green), total VCP (red) and DAPI DNA counterstain (blue) of wild-type and Vcpkmt-/-. Scale bars 20 μm. (A) Cultured mouse embryonic fibroblast (MEFs). (B) Semniferous tubuli of testes. (C) Hepatocytes with a central vein. (D) Kidney cortex.

Mentions: Next we wanted to test if VCP is methylated in vivo and if this methylation is mediated solely by VCPKMT or if maybe other methyltransferases could compensate the loss of VCPKMT in mice. We immunoprecipitated total VCP from different mouse tissues, ran it on an SDS-PAGE-gel, followed by excision of the relevant band, in-gel digestion with Arg-C, and analysis of the resulting peptides by mass spectrometry (Fig 2A). The presence of VCPKMT in wild-type mice results in almost exclusively trimethylated VCP, while only about 0.5% of the VCP molecules are dimethylated. In the four Vcpkmt-/- tissues analyzed neither mono-, di- or trimethylated VCP could be detected (Fig 2A right panel). To further study the VCP-K315 methylation status in different tissues we generated a K315me3-VCP specific antibody, which only recognizes a trimethylated peptide containing the sequence around VCP-K315 but not the matching unmodified sequence (S3 Fig). We then used this K315me3-VCP antibody to analyze by Western blotting the VCP methylation in whole cell protein extracts from various tissues. In all the organs examined, the methylation signal was present in the wild-type, but completely absent in the extracts from Vcpkmt-/- mice (Fig 2B). An antibody recognizing VCP, regardless of its methylation status, was used as a loading control and VCP extent did not vary between wild-type and Vcpkmt-/- mice (Fig 2B). To further establish the distribution of K315me3 in vivo, we generated primary MEFs and analyzed K315me3 by immunofluorescence staining (Fig 3A). K315me3 could only be detected in the wild-type and showed an almost perfect co-localization with the total VCP. In addition we investigated the methylation status of VCP in mouse tissues. For that purpose we used co-immunostaining of VCP and K315me3-VCP in formalin fixed, paraffin embedded tissue sections. In testis (Fig 3B), liver (Fig 3C) and kidney (Fig 3D) it is clearly visible, that in Vcpkmt-/- mice the trimethylation signal is severely diminished. This difference is not as striking as in the primary MEFs, probably because of the different fixation methods. In addition we analyzed the K315me3 distribution in spleen and lung, which gave comparable results (S4 Fig).


Lysine Methylation of the Valosin-Containing Protein (VCP) Is Dispensable for Development and Survival of Mice.

Fusser M, Kernstock S, Aileni VK, Egge-Jacobsen W, Falnes PØ, Klungland A - PLoS ONE (2015)

Immunostaining of K315me3-VCP in cells and tissues of wild-type and Vcpkmt-/- mice.Immunostaining of K315me3-VCP (green), total VCP (red) and DAPI DNA counterstain (blue) of wild-type and Vcpkmt-/-. Scale bars 20 μm. (A) Cultured mouse embryonic fibroblast (MEFs). (B) Semniferous tubuli of testes. (C) Hepatocytes with a central vein. (D) Kidney cortex.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4636187&req=5

pone.0141472.g003: Immunostaining of K315me3-VCP in cells and tissues of wild-type and Vcpkmt-/- mice.Immunostaining of K315me3-VCP (green), total VCP (red) and DAPI DNA counterstain (blue) of wild-type and Vcpkmt-/-. Scale bars 20 μm. (A) Cultured mouse embryonic fibroblast (MEFs). (B) Semniferous tubuli of testes. (C) Hepatocytes with a central vein. (D) Kidney cortex.
Mentions: Next we wanted to test if VCP is methylated in vivo and if this methylation is mediated solely by VCPKMT or if maybe other methyltransferases could compensate the loss of VCPKMT in mice. We immunoprecipitated total VCP from different mouse tissues, ran it on an SDS-PAGE-gel, followed by excision of the relevant band, in-gel digestion with Arg-C, and analysis of the resulting peptides by mass spectrometry (Fig 2A). The presence of VCPKMT in wild-type mice results in almost exclusively trimethylated VCP, while only about 0.5% of the VCP molecules are dimethylated. In the four Vcpkmt-/- tissues analyzed neither mono-, di- or trimethylated VCP could be detected (Fig 2A right panel). To further study the VCP-K315 methylation status in different tissues we generated a K315me3-VCP specific antibody, which only recognizes a trimethylated peptide containing the sequence around VCP-K315 but not the matching unmodified sequence (S3 Fig). We then used this K315me3-VCP antibody to analyze by Western blotting the VCP methylation in whole cell protein extracts from various tissues. In all the organs examined, the methylation signal was present in the wild-type, but completely absent in the extracts from Vcpkmt-/- mice (Fig 2B). An antibody recognizing VCP, regardless of its methylation status, was used as a loading control and VCP extent did not vary between wild-type and Vcpkmt-/- mice (Fig 2B). To further establish the distribution of K315me3 in vivo, we generated primary MEFs and analyzed K315me3 by immunofluorescence staining (Fig 3A). K315me3 could only be detected in the wild-type and showed an almost perfect co-localization with the total VCP. In addition we investigated the methylation status of VCP in mouse tissues. For that purpose we used co-immunostaining of VCP and K315me3-VCP in formalin fixed, paraffin embedded tissue sections. In testis (Fig 3B), liver (Fig 3C) and kidney (Fig 3D) it is clearly visible, that in Vcpkmt-/- mice the trimethylation signal is severely diminished. This difference is not as striking as in the primary MEFs, probably because of the different fixation methods. In addition we analyzed the K315me3 distribution in spleen and lung, which gave comparable results (S4 Fig).

Bottom Line: We show that Vcpkmt is ubiquitously expressed in all tissues examined and confirm the sub-cellular localization to the cytoplasm.The results show that VCPKMT is a highly specific enzyme, and suggest that VCP is its sole substrate.Overall the results show that VCPKMT is an enzyme required for methylation of K315 of VCP in vivo, but VCPKMT is not essential for development or survival under unstressed conditions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Oslo University Hospital, Rikshospitalet, Oslo, Norway.

ABSTRACT
Valosin-containing protein (VCP) is a homohexameric ATPase involved in a multitude cellular processes and it was recently shown that VCP is trimethylated at lysine 315 by the VCP lysine methyltransferase (VCPKMT). Here, we generated and validated a constitutive knockout mouse by targeting exon 1-4 of the Vcpkmt gene. We show that Vcpkmt is ubiquitously expressed in all tissues examined and confirm the sub-cellular localization to the cytoplasm. We show by (I) mass spectrometric analysis, (II) VCPKMT-mediated in vitro methylation of VCP in cell extracts and (III) immunostaining with a methylation specific antibody, that in Vcpkmt-/- mice the methylation of lysine 315 in VCP is completely abolished. In contrast, VCP is almost exclusively trimethylated in wild-type mice. Furthermore, we investigated the specificity of VCPKMT with in vitro methylation assays using as source of substrate protein extracts from Vcpkmt-/- mouse organs or three human Vcpkmt-/- cell lines. The results show that VCPKMT is a highly specific enzyme, and suggest that VCP is its sole substrate. The Vcpkmt-/- mice were viable, fertile and had no obvious pathological phenotype. Their body weight, life span and acute endurance capacity were comparable to wild-type controls. Overall the results show that VCPKMT is an enzyme required for methylation of K315 of VCP in vivo, but VCPKMT is not essential for development or survival under unstressed conditions.

No MeSH data available.


Related in: MedlinePlus