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Mastermind-Like 1 Is Ubiquitinated: Functional Consequences for Notch Signaling.

Farshbaf M, Lindberg MJ, Truong A, Bevens Z, Chambers E, Pournara A, Wallberg AE, White JB - PLoS ONE (2015)

Bottom Line: We also show that p300 stimulates ubiquitination and that Notch inhibits ubiquitination.Therefore, we speculate that ubiquitination of MAML1 might be a mechanism to maintain low levels of the protein until needed for transcriptional activation.Our data supports the notion that a precise and tight regulation of the Notch pathway is required for this signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Biological Sciences, San José State University, San José, California, United States of America.

ABSTRACT
Early studies demonstrated the involvement of ubiquitination of the Notch intracellular domain for rapid turnover of the transcriptional complex at Notch target genes. It was shown that this ubiquitination was promoted by the co-activator Mastermind like 1 (MAML1). MAML1 also contains numerous lysine residues that may also be ubiquitinated and necessary for protein regulation. In this study, we show that over-expressed MAML1 is ubiquitinated and identify eight conserved lysine residues which are required for ubiquitination. We also show that p300 stimulates ubiquitination and that Notch inhibits ubiquitination. Furthermore, we show that a mutant MAML1 that has decreased ubiquitination shows increased output from a HES1 reporter gene assay. Therefore, we speculate that ubiquitination of MAML1 might be a mechanism to maintain low levels of the protein until needed for transcriptional activation. In summary, this study identifies that MAML1 is ubiquitinated in the absence of Notch signaling to maintain low levels of MAML1 in the cell. Our data supports the notion that a precise and tight regulation of the Notch pathway is required for this signaling pathway.

No MeSH data available.


Related in: MedlinePlus

MAML1 ubiquitination is stimulated by p300 and inhibited by NICD.(A, B, C) Western blot analysis for ubiquitination of myc-tagged MAML1 or MAML1-300 was performed in the presence or absence of p300 (6A and 6B) or N1ICD (6C). MAML1 proteins and HA-Ub were expressed and immunoprecipitated as described in the text in the presence or absence of p300 or NICD. Prior to IP, a sample was taken to monitor total protein levels for MAML1. Western blots were performed to either the IP for ubiquitination or total extract was blotted as a loading control for MAML1 expression. p300 stimulated the ubiquitination of both MAML1 and MAML1 1–300 (compare lanes 2 and 3 in 6A and 6B) whereas NICD decreased the amount of ubiquitination of MAML1 (compare lanes 3 and 4 in 6C). p300 and p300ΔHAT stimulated degradation of MAML1 (6D and 6E). MAML1 was overexpressed with p300 or p300ΔHAT and pulse-chase experiments performed as previously described (See Fig 2 text). Western blots were performed and results were normalized to GAPDH levels and results are shown ± SD (n = 4). (6F) The half-life of MAML1 is significantly decreased in the presence of p300 (p = 0.025) or p300ΔHAT (p = 0.0146).
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pone.0134013.g006: MAML1 ubiquitination is stimulated by p300 and inhibited by NICD.(A, B, C) Western blot analysis for ubiquitination of myc-tagged MAML1 or MAML1-300 was performed in the presence or absence of p300 (6A and 6B) or N1ICD (6C). MAML1 proteins and HA-Ub were expressed and immunoprecipitated as described in the text in the presence or absence of p300 or NICD. Prior to IP, a sample was taken to monitor total protein levels for MAML1. Western blots were performed to either the IP for ubiquitination or total extract was blotted as a loading control for MAML1 expression. p300 stimulated the ubiquitination of both MAML1 and MAML1 1–300 (compare lanes 2 and 3 in 6A and 6B) whereas NICD decreased the amount of ubiquitination of MAML1 (compare lanes 3 and 4 in 6C). p300 and p300ΔHAT stimulated degradation of MAML1 (6D and 6E). MAML1 was overexpressed with p300 or p300ΔHAT and pulse-chase experiments performed as previously described (See Fig 2 text). Western blots were performed and results were normalized to GAPDH levels and results are shown ± SD (n = 4). (6F) The half-life of MAML1 is significantly decreased in the presence of p300 (p = 0.025) or p300ΔHAT (p = 0.0146).

Mentions: We next conducted biochemical experiments to look at how expression of other protein binding partners would affect the ubiquitination of MAML1. Overexpression of Myc-tagged MAML1 with Flag-tagged p300 and HA-Ub resulted in an increased ubiquitination of MAML1 compared to MAML1 and HA-Ub alone (Fig 6A compare Lanes 2 and 3). The p300 stimulated ubiquitination also occurred with the MAML1 1–300 construct (Fig 6B compare lanes 2 and 3). Interestingly, overexpression of MAML1 with NICD resulted in a decrease in the ubiquitination of MAML1 (Fig 6C, compare lanes) suggesting that the interaction with the NICD blocks the ability of MAML1 to be ubiquitinated.


Mastermind-Like 1 Is Ubiquitinated: Functional Consequences for Notch Signaling.

Farshbaf M, Lindberg MJ, Truong A, Bevens Z, Chambers E, Pournara A, Wallberg AE, White JB - PLoS ONE (2015)

MAML1 ubiquitination is stimulated by p300 and inhibited by NICD.(A, B, C) Western blot analysis for ubiquitination of myc-tagged MAML1 or MAML1-300 was performed in the presence or absence of p300 (6A and 6B) or N1ICD (6C). MAML1 proteins and HA-Ub were expressed and immunoprecipitated as described in the text in the presence or absence of p300 or NICD. Prior to IP, a sample was taken to monitor total protein levels for MAML1. Western blots were performed to either the IP for ubiquitination or total extract was blotted as a loading control for MAML1 expression. p300 stimulated the ubiquitination of both MAML1 and MAML1 1–300 (compare lanes 2 and 3 in 6A and 6B) whereas NICD decreased the amount of ubiquitination of MAML1 (compare lanes 3 and 4 in 6C). p300 and p300ΔHAT stimulated degradation of MAML1 (6D and 6E). MAML1 was overexpressed with p300 or p300ΔHAT and pulse-chase experiments performed as previously described (See Fig 2 text). Western blots were performed and results were normalized to GAPDH levels and results are shown ± SD (n = 4). (6F) The half-life of MAML1 is significantly decreased in the presence of p300 (p = 0.025) or p300ΔHAT (p = 0.0146).
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pone.0134013.g006: MAML1 ubiquitination is stimulated by p300 and inhibited by NICD.(A, B, C) Western blot analysis for ubiquitination of myc-tagged MAML1 or MAML1-300 was performed in the presence or absence of p300 (6A and 6B) or N1ICD (6C). MAML1 proteins and HA-Ub were expressed and immunoprecipitated as described in the text in the presence or absence of p300 or NICD. Prior to IP, a sample was taken to monitor total protein levels for MAML1. Western blots were performed to either the IP for ubiquitination or total extract was blotted as a loading control for MAML1 expression. p300 stimulated the ubiquitination of both MAML1 and MAML1 1–300 (compare lanes 2 and 3 in 6A and 6B) whereas NICD decreased the amount of ubiquitination of MAML1 (compare lanes 3 and 4 in 6C). p300 and p300ΔHAT stimulated degradation of MAML1 (6D and 6E). MAML1 was overexpressed with p300 or p300ΔHAT and pulse-chase experiments performed as previously described (See Fig 2 text). Western blots were performed and results were normalized to GAPDH levels and results are shown ± SD (n = 4). (6F) The half-life of MAML1 is significantly decreased in the presence of p300 (p = 0.025) or p300ΔHAT (p = 0.0146).
Mentions: We next conducted biochemical experiments to look at how expression of other protein binding partners would affect the ubiquitination of MAML1. Overexpression of Myc-tagged MAML1 with Flag-tagged p300 and HA-Ub resulted in an increased ubiquitination of MAML1 compared to MAML1 and HA-Ub alone (Fig 6A compare Lanes 2 and 3). The p300 stimulated ubiquitination also occurred with the MAML1 1–300 construct (Fig 6B compare lanes 2 and 3). Interestingly, overexpression of MAML1 with NICD resulted in a decrease in the ubiquitination of MAML1 (Fig 6C, compare lanes) suggesting that the interaction with the NICD blocks the ability of MAML1 to be ubiquitinated.

Bottom Line: We also show that p300 stimulates ubiquitination and that Notch inhibits ubiquitination.Therefore, we speculate that ubiquitination of MAML1 might be a mechanism to maintain low levels of the protein until needed for transcriptional activation.Our data supports the notion that a precise and tight regulation of the Notch pathway is required for this signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Biological Sciences, San José State University, San José, California, United States of America.

ABSTRACT
Early studies demonstrated the involvement of ubiquitination of the Notch intracellular domain for rapid turnover of the transcriptional complex at Notch target genes. It was shown that this ubiquitination was promoted by the co-activator Mastermind like 1 (MAML1). MAML1 also contains numerous lysine residues that may also be ubiquitinated and necessary for protein regulation. In this study, we show that over-expressed MAML1 is ubiquitinated and identify eight conserved lysine residues which are required for ubiquitination. We also show that p300 stimulates ubiquitination and that Notch inhibits ubiquitination. Furthermore, we show that a mutant MAML1 that has decreased ubiquitination shows increased output from a HES1 reporter gene assay. Therefore, we speculate that ubiquitination of MAML1 might be a mechanism to maintain low levels of the protein until needed for transcriptional activation. In summary, this study identifies that MAML1 is ubiquitinated in the absence of Notch signaling to maintain low levels of MAML1 in the cell. Our data supports the notion that a precise and tight regulation of the Notch pathway is required for this signaling pathway.

No MeSH data available.


Related in: MedlinePlus