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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

Generation and validation of Vcpkmt knockout.(A) Schematic overview of K315 trimethylation of VCP monomer by VCPKMT. AdoMet = S-adenosyl methionine (B) Targeting of the Vcpkmt gene. Diagram shows the endogenous murine Vcpkmt locus, the position of the targeted gene and the recombined gene. (C) Representative genotyping result from Vcpkmt wild-type (+/+), knockout (-/-) and heterozygous (+/-) mice. (D) Immunostaining for VCPKMT in wild-type and Vcpkmt-/- mouse embryonic fibroblasts. DNA counterstain with DAPI. Scale bars 10 μm. (E) The gene expression of Vcpkmt relative to Gapdh in various murine tissues measured with quantitative RT-PCR. n = 3 mice, means ± SD.
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pone.0141472.g001: Generation and validation of Vcpkmt knockout.(A) Schematic overview of K315 trimethylation of VCP monomer by VCPKMT. AdoMet = S-adenosyl methionine (B) Targeting of the Vcpkmt gene. Diagram shows the endogenous murine Vcpkmt locus, the position of the targeted gene and the recombined gene. (C) Representative genotyping result from Vcpkmt wild-type (+/+), knockout (-/-) and heterozygous (+/-) mice. (D) Immunostaining for VCPKMT in wild-type and Vcpkmt-/- mouse embryonic fibroblasts. DNA counterstain with DAPI. Scale bars 10 μm. (E) The gene expression of Vcpkmt relative to Gapdh in various murine tissues measured with quantitative RT-PCR. n = 3 mice, means ± SD.

Mentions: VCPKMT is a MT that uses AdoMet to trimethylate K315 in the VCP monomer. Trimethylated VCPs are then assembled to a fully methylated homohexameric complex (Fig 1A). We generated a conditional knockout mouse for Vcpkmt by introducing loxP sites flanking exons 1 and 4 of the Vcpkmt gene (Fig 1B) and thereby deleting more than 82% of the Vcpkmt coding region. After breeding these recombined mice with constitutively Cre recombinase-expressing mice, complete removal of genome sequences spanning exon 1 to 4 of Vcpkmt was confirmed. The DNA from all offspring was analyzed by PCR with specific primers, as outlined in the methods section, to determine their genotype (Fig 1C and S1 Fig). We generated mouse embryonic fibroblasts (MEFs) from wild-type and Vcpkmt-/- mice. Cells lacking VCPKMT had proliferation rates and growth characteristics comparable to wild-type cells (S2 Fig). VCPKMT depletion in Vcpkmt-/- mice was verified with immunofluorescence with VCPKMT anti-serum (Fig 1D). In the wild-type MEFs VCPKMT is predominantly localized to the cytoplasm (Fig 1D).


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)

Generation and validation of Vcpkmt knockout.(A) Schematic overview of K315 trimethylation of VCP monomer by VCPKMT. AdoMet = S-adenosyl methionine (B) Targeting of the Vcpkmt gene. Diagram shows the endogenous murine Vcpkmt locus, the position of the targeted gene and the recombined gene. (C) Representative genotyping result from Vcpkmt wild-type (+/+), knockout (-/-) and heterozygous (+/-) mice. (D) Immunostaining for VCPKMT in wild-type and Vcpkmt-/- mouse embryonic fibroblasts. DNA counterstain with DAPI. Scale bars 10 μm. (E) The gene expression of Vcpkmt relative to Gapdh in various murine tissues measured with quantitative RT-PCR. n = 3 mice, means ± SD.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4636187&req=5

pone.0141472.g001: Generation and validation of Vcpkmt knockout.(A) Schematic overview of K315 trimethylation of VCP monomer by VCPKMT. AdoMet = S-adenosyl methionine (B) Targeting of the Vcpkmt gene. Diagram shows the endogenous murine Vcpkmt locus, the position of the targeted gene and the recombined gene. (C) Representative genotyping result from Vcpkmt wild-type (+/+), knockout (-/-) and heterozygous (+/-) mice. (D) Immunostaining for VCPKMT in wild-type and Vcpkmt-/- mouse embryonic fibroblasts. DNA counterstain with DAPI. Scale bars 10 μm. (E) The gene expression of Vcpkmt relative to Gapdh in various murine tissues measured with quantitative RT-PCR. n = 3 mice, means ± SD.
Mentions: VCPKMT is a MT that uses AdoMet to trimethylate K315 in the VCP monomer. Trimethylated VCPs are then assembled to a fully methylated homohexameric complex (Fig 1A). We generated a conditional knockout mouse for Vcpkmt by introducing loxP sites flanking exons 1 and 4 of the Vcpkmt gene (Fig 1B) and thereby deleting more than 82% of the Vcpkmt coding region. After breeding these recombined mice with constitutively Cre recombinase-expressing mice, complete removal of genome sequences spanning exon 1 to 4 of Vcpkmt was confirmed. The DNA from all offspring was analyzed by PCR with specific primers, as outlined in the methods section, to determine their genotype (Fig 1C and S1 Fig). We generated mouse embryonic fibroblasts (MEFs) from wild-type and Vcpkmt-/- mice. Cells lacking VCPKMT had proliferation rates and growth characteristics comparable to wild-type cells (S2 Fig). VCPKMT depletion in Vcpkmt-/- mice was verified with immunofluorescence with VCPKMT anti-serum (Fig 1D). In the wild-type MEFs VCPKMT is predominantly localized to the cytoplasm (Fig 1D).

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