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The menin tumor suppressor protein is phosphorylated in response to DNA damage.

Francis J, Lin W, Rozenblatt-Rosen O, Meyerson M - PLoS ONE (2011)

Bottom Line: Multiple endocrine neoplasia type 1 (MEN1) is a heritable cancer syndrome characterized by tumors of the pituitary, pancreas and parathyroid.Phosphorylation at these sites however does not influence the ability to interact with histone methyltransferase activity.In contrast, the interaction between menin and RNA polymerase II is influenced by phosphorylation, whereby a phospho-deficient mutant had a higher affinity for the elongating form of RNA polymerase compared to wild type.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: Multiple endocrine neoplasia type 1 (MEN1) is a heritable cancer syndrome characterized by tumors of the pituitary, pancreas and parathyroid. Menin, the product of the MEN1 gene, is a tumor suppressor protein that functions in part through the regulation of transcription mediated by interactions with chromatin modifying enzymes.

Principal findings: Here we show menin association with the 5' regions of DNA damage response genes increases after DNA damage and is correlated with RNA polymerase II association but not with changes in histone methylation. Furthermore, we were able to detect significant levels of menin at the 3' regions of CDKN1A and GADD45A under conditions of enhanced transcription following DNA damage. We also demonstrate that menin is specifically phosphorylated at Ser394 in response to several forms of DNA damage, Ser487 is dynamically phosphorylated and Ser543 is constitutively phosphorylated. Phosphorylation at these sites however does not influence the ability to interact with histone methyltransferase activity. In contrast, the interaction between menin and RNA polymerase II is influenced by phosphorylation, whereby a phospho-deficient mutant had a higher affinity for the elongating form of RNA polymerase compared to wild type. Additionally, a subset of MEN1-associated missense point mutants, fail to undergo DNA damage dependent phosphorylation.

Conclusion: Together, our findings suggest that the menin tumor suppressor protein undergoes DNA damage induced phosphorylation and participates in the DNA damage transcriptional response.

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Related in: MedlinePlus

MEN1 missense point mutants display altered phosphorylation.(A) Flag IPs from293T cells transfected with Flag-wildtype menin, or Flag missense mutants following treatment with 0.05uM Adr for 18h, or 30 minutes after 25J/m2 UV. IPs were immunoblotted with phospho-Ser394, phospho-Ser487, phospho-Ser543 or total menin. (B) Menin IPs from Men1−/− MEFs expressing wildtype or phospho-deficient point mutants were incubated with histone H3 and the methyl donor 3H-SAM to assay for histone methyltransferase activity. Reactions were resolved on 15% SDS-PAGE, amplified, dried and fluorographed. (C) Flag IPs from 293T cells transfected with phospho-deficient point mutants 30 minutes following treatment with 25 J/m2 UV or 18 h following 0.05 uM Adr, were resolved and immunoblotted with phospho-menin antibodies or phospho-RNAPII antibodies.
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pone-0016119-g006: MEN1 missense point mutants display altered phosphorylation.(A) Flag IPs from293T cells transfected with Flag-wildtype menin, or Flag missense mutants following treatment with 0.05uM Adr for 18h, or 30 minutes after 25J/m2 UV. IPs were immunoblotted with phospho-Ser394, phospho-Ser487, phospho-Ser543 or total menin. (B) Menin IPs from Men1−/− MEFs expressing wildtype or phospho-deficient point mutants were incubated with histone H3 and the methyl donor 3H-SAM to assay for histone methyltransferase activity. Reactions were resolved on 15% SDS-PAGE, amplified, dried and fluorographed. (C) Flag IPs from 293T cells transfected with phospho-deficient point mutants 30 minutes following treatment with 25 J/m2 UV or 18 h following 0.05 uM Adr, were resolved and immunoblotted with phospho-menin antibodies or phospho-RNAPII antibodies.

Mentions: A major question in the menin field is the link between missense point mutations and their loss of various menin functions— in particular, some but not all of these mutants lack the ability to interact with KMT2A/KMT2D [12]. To determine whether or not MEN1-associated missense point mutants are phosphorylated, we screened a panel of mutants for the ability to undergo Ser487, Ser543 and Ser394 phosphorylation. Flag-tagged point mutants were immunoprecipitated from transiently transfected 293T cells following treatment with Adr or UV. As shown in Figure 6A, the mutants P12L, L22R, and A309P fail to achieve the level of Ser394 phosphorylation that is observed in the wild type menin and the H139D and A242Vmutants. In addition, the P12L mutant also displayed altered Ser487 phosphorylation following UV treatment (Figure 6A).


The menin tumor suppressor protein is phosphorylated in response to DNA damage.

Francis J, Lin W, Rozenblatt-Rosen O, Meyerson M - PLoS ONE (2011)

MEN1 missense point mutants display altered phosphorylation.(A) Flag IPs from293T cells transfected with Flag-wildtype menin, or Flag missense mutants following treatment with 0.05uM Adr for 18h, or 30 minutes after 25J/m2 UV. IPs were immunoblotted with phospho-Ser394, phospho-Ser487, phospho-Ser543 or total menin. (B) Menin IPs from Men1−/− MEFs expressing wildtype or phospho-deficient point mutants were incubated with histone H3 and the methyl donor 3H-SAM to assay for histone methyltransferase activity. Reactions were resolved on 15% SDS-PAGE, amplified, dried and fluorographed. (C) Flag IPs from 293T cells transfected with phospho-deficient point mutants 30 minutes following treatment with 25 J/m2 UV or 18 h following 0.05 uM Adr, were resolved and immunoblotted with phospho-menin antibodies or phospho-RNAPII antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3021530&req=5

pone-0016119-g006: MEN1 missense point mutants display altered phosphorylation.(A) Flag IPs from293T cells transfected with Flag-wildtype menin, or Flag missense mutants following treatment with 0.05uM Adr for 18h, or 30 minutes after 25J/m2 UV. IPs were immunoblotted with phospho-Ser394, phospho-Ser487, phospho-Ser543 or total menin. (B) Menin IPs from Men1−/− MEFs expressing wildtype or phospho-deficient point mutants were incubated with histone H3 and the methyl donor 3H-SAM to assay for histone methyltransferase activity. Reactions were resolved on 15% SDS-PAGE, amplified, dried and fluorographed. (C) Flag IPs from 293T cells transfected with phospho-deficient point mutants 30 minutes following treatment with 25 J/m2 UV or 18 h following 0.05 uM Adr, were resolved and immunoblotted with phospho-menin antibodies or phospho-RNAPII antibodies.
Mentions: A major question in the menin field is the link between missense point mutations and their loss of various menin functions— in particular, some but not all of these mutants lack the ability to interact with KMT2A/KMT2D [12]. To determine whether or not MEN1-associated missense point mutants are phosphorylated, we screened a panel of mutants for the ability to undergo Ser487, Ser543 and Ser394 phosphorylation. Flag-tagged point mutants were immunoprecipitated from transiently transfected 293T cells following treatment with Adr or UV. As shown in Figure 6A, the mutants P12L, L22R, and A309P fail to achieve the level of Ser394 phosphorylation that is observed in the wild type menin and the H139D and A242Vmutants. In addition, the P12L mutant also displayed altered Ser487 phosphorylation following UV treatment (Figure 6A).

Bottom Line: Multiple endocrine neoplasia type 1 (MEN1) is a heritable cancer syndrome characterized by tumors of the pituitary, pancreas and parathyroid.Phosphorylation at these sites however does not influence the ability to interact with histone methyltransferase activity.In contrast, the interaction between menin and RNA polymerase II is influenced by phosphorylation, whereby a phospho-deficient mutant had a higher affinity for the elongating form of RNA polymerase compared to wild type.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: Multiple endocrine neoplasia type 1 (MEN1) is a heritable cancer syndrome characterized by tumors of the pituitary, pancreas and parathyroid. Menin, the product of the MEN1 gene, is a tumor suppressor protein that functions in part through the regulation of transcription mediated by interactions with chromatin modifying enzymes.

Principal findings: Here we show menin association with the 5' regions of DNA damage response genes increases after DNA damage and is correlated with RNA polymerase II association but not with changes in histone methylation. Furthermore, we were able to detect significant levels of menin at the 3' regions of CDKN1A and GADD45A under conditions of enhanced transcription following DNA damage. We also demonstrate that menin is specifically phosphorylated at Ser394 in response to several forms of DNA damage, Ser487 is dynamically phosphorylated and Ser543 is constitutively phosphorylated. Phosphorylation at these sites however does not influence the ability to interact with histone methyltransferase activity. In contrast, the interaction between menin and RNA polymerase II is influenced by phosphorylation, whereby a phospho-deficient mutant had a higher affinity for the elongating form of RNA polymerase compared to wild type. Additionally, a subset of MEN1-associated missense point mutants, fail to undergo DNA damage dependent phosphorylation.

Conclusion: Together, our findings suggest that the menin tumor suppressor protein undergoes DNA damage induced phosphorylation and participates in the DNA damage transcriptional response.

Show MeSH
Related in: MedlinePlus