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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

Menin phosphorylation is dependent upon ATM and ATR.(A) Menin IPs from control GM03323, ATM deficient GM03189 and ATR deficient GM18326 lymphoblastoid cells were performed 90 minutes after exposure to 1000 Rads γ-IR or 30 minutes after 25J/m2 UV. IPs were resolved and immunoblotted with phospho-Ser394 or total menin antibodies. Whole cell extracts from each cell type were immunoblotted with phospho-Ser15-p53 and total p53 (lower panel). (B) 293T cells were pretreated with 10 uM KU55933 for 2 hours prior to treatment with 1000 Rads γ-IR or 25J/m2 UV. Menin IPs were performed, resolved and immunoblotted for phospho-Ser394 or total menin. The same whole cell extracts were immunoblotted with phospho-Ser15-p53 and total p53 (lower panel). (C) 293T cells were pretreated with 10 uM DNA-PK inhibitor II for 2 hours prior to treatment with 1000 Rads γ-IR or 25J/m2 UV. Menin IPs were performed, resolved and immunoblotted for phospho-Ser394 or total menin. The same whole cell extracts were immunoblotted with phospho-Ser15-p53 and total p53 (lower panel).
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pone-0016119-g005: Menin phosphorylation is dependent upon ATM and ATR.(A) Menin IPs from control GM03323, ATM deficient GM03189 and ATR deficient GM18326 lymphoblastoid cells were performed 90 minutes after exposure to 1000 Rads γ-IR or 30 minutes after 25J/m2 UV. IPs were resolved and immunoblotted with phospho-Ser394 or total menin antibodies. Whole cell extracts from each cell type were immunoblotted with phospho-Ser15-p53 and total p53 (lower panel). (B) 293T cells were pretreated with 10 uM KU55933 for 2 hours prior to treatment with 1000 Rads γ-IR or 25J/m2 UV. Menin IPs were performed, resolved and immunoblotted for phospho-Ser394 or total menin. The same whole cell extracts were immunoblotted with phospho-Ser15-p53 and total p53 (lower panel). (C) 293T cells were pretreated with 10 uM DNA-PK inhibitor II for 2 hours prior to treatment with 1000 Rads γ-IR or 25J/m2 UV. Menin IPs were performed, resolved and immunoblotted for phospho-Ser394 or total menin. The same whole cell extracts were immunoblotted with phospho-Ser15-p53 and total p53 (lower panel).

Mentions: The DNA damage transducing kinases ATM, ATM- and Rad3-related (ATR) and DNA-Protein Kinase (DNA-PK) are activated after damage and phosphorylate downstream targets containing SQ/TQ recognition motifs. To determine whether menin Ser394 phosphorylation was dependent upon ATM and/or ATR following DNA damage we utilized cells deficient in these enzymes. Following γ-IR treatment, there was no detectable phospho-Ser394 in ATM-deficient GM03189 cells while a strong signal was detected in the ATR-deficient GM18326 cells and control GM03323 cells (Figure 5A, upper panel, lanes 2, 5 and 8). In contrast, UV treatment resulted in a strong induction of Ser394 phosphorylation only in control cells while very weak signals were seen in both ATM-and ATR-deficient cells. Phosphorylation of p53 at Ser15 was also abrogated in the ATM deficient cell line after γ-IR and UV, indicating the importance of ATM signaling after both forms of damage (Figure 5A, lower panel).


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

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

Menin phosphorylation is dependent upon ATM and ATR.(A) Menin IPs from control GM03323, ATM deficient GM03189 and ATR deficient GM18326 lymphoblastoid cells were performed 90 minutes after exposure to 1000 Rads γ-IR or 30 minutes after 25J/m2 UV. IPs were resolved and immunoblotted with phospho-Ser394 or total menin antibodies. Whole cell extracts from each cell type were immunoblotted with phospho-Ser15-p53 and total p53 (lower panel). (B) 293T cells were pretreated with 10 uM KU55933 for 2 hours prior to treatment with 1000 Rads γ-IR or 25J/m2 UV. Menin IPs were performed, resolved and immunoblotted for phospho-Ser394 or total menin. The same whole cell extracts were immunoblotted with phospho-Ser15-p53 and total p53 (lower panel). (C) 293T cells were pretreated with 10 uM DNA-PK inhibitor II for 2 hours prior to treatment with 1000 Rads γ-IR or 25J/m2 UV. Menin IPs were performed, resolved and immunoblotted for phospho-Ser394 or total menin. The same whole cell extracts were immunoblotted with phospho-Ser15-p53 and total p53 (lower panel).
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Related In: Results  -  Collection

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pone-0016119-g005: Menin phosphorylation is dependent upon ATM and ATR.(A) Menin IPs from control GM03323, ATM deficient GM03189 and ATR deficient GM18326 lymphoblastoid cells were performed 90 minutes after exposure to 1000 Rads γ-IR or 30 minutes after 25J/m2 UV. IPs were resolved and immunoblotted with phospho-Ser394 or total menin antibodies. Whole cell extracts from each cell type were immunoblotted with phospho-Ser15-p53 and total p53 (lower panel). (B) 293T cells were pretreated with 10 uM KU55933 for 2 hours prior to treatment with 1000 Rads γ-IR or 25J/m2 UV. Menin IPs were performed, resolved and immunoblotted for phospho-Ser394 or total menin. The same whole cell extracts were immunoblotted with phospho-Ser15-p53 and total p53 (lower panel). (C) 293T cells were pretreated with 10 uM DNA-PK inhibitor II for 2 hours prior to treatment with 1000 Rads γ-IR or 25J/m2 UV. Menin IPs were performed, resolved and immunoblotted for phospho-Ser394 or total menin. The same whole cell extracts were immunoblotted with phospho-Ser15-p53 and total p53 (lower panel).
Mentions: The DNA damage transducing kinases ATM, ATM- and Rad3-related (ATR) and DNA-Protein Kinase (DNA-PK) are activated after damage and phosphorylate downstream targets containing SQ/TQ recognition motifs. To determine whether menin Ser394 phosphorylation was dependent upon ATM and/or ATR following DNA damage we utilized cells deficient in these enzymes. Following γ-IR treatment, there was no detectable phospho-Ser394 in ATM-deficient GM03189 cells while a strong signal was detected in the ATR-deficient GM18326 cells and control GM03323 cells (Figure 5A, upper panel, lanes 2, 5 and 8). In contrast, UV treatment resulted in a strong induction of Ser394 phosphorylation only in control cells while very weak signals were seen in both ATM-and ATR-deficient cells. Phosphorylation of p53 at Ser15 was also abrogated in the ATM deficient cell line after γ-IR and UV, indicating the importance of ATM signaling after both forms of damage (Figure 5A, lower panel).

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