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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

Half-life studies to MAML1-3 family members.(3A) MAML1, MAML1Δ75–301, MAML1-1-300, MAML2 or MAML3 were expressed with myc-tagged CBF1 in HeLa cells. Cells were treated with 150 μg/ml cycloheximide and cell extracts collected every hour for 5 hours. Western blots were performed to the myc-tag for the various proteins or to endogenously expressed GAPDH. Results shown are from representative blots form three different experiments. Black and gray arrowheads indicate degradation products that could be detected with time with the MAML1 and MAML1-300 constructs. (3B) ImageJ software was used to quantitate expression levels normalized to either CBF1 or GAPDH. The data is presented as the mean ± SD in bar graphs. A * indicates significantly different compared to MAML1 (p<0.05).
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pone.0134013.g003: Half-life studies to MAML1-3 family members.(3A) MAML1, MAML1Δ75–301, MAML1-1-300, MAML2 or MAML3 were expressed with myc-tagged CBF1 in HeLa cells. Cells were treated with 150 μg/ml cycloheximide and cell extracts collected every hour for 5 hours. Western blots were performed to the myc-tag for the various proteins or to endogenously expressed GAPDH. Results shown are from representative blots form three different experiments. Black and gray arrowheads indicate degradation products that could be detected with time with the MAML1 and MAML1-300 constructs. (3B) ImageJ software was used to quantitate expression levels normalized to either CBF1 or GAPDH. The data is presented as the mean ± SD in bar graphs. A * indicates significantly different compared to MAML1 (p<0.05).

Mentions: We have also conducted pulse chase experiments to determine the half-life of MAML1, MAML1 1–300, and MAML1Δ75–301. Myc-tagged vectors for the different constructs were overexpressed with myc-tagged CBF1 in HeLa cells. Representative blots are shown in Fig 3A of the results of the pulse-chase studies conducted for the different MAML1 proteins. Over time, an increase in a degradation product was detected for MAML1. The black arrowhead shows a degradation product that is immediately detected at the first time point and increases over time with maximum amounts seen between 2 and 3 hours. The gray arrowhead shows a degradation product that is seen early, but only begins to increase in amount after the three hour time point. This same trend could also be seen for the MAML1-1-300. The MAML1-1-300 is detected as a doublet as indicated by the black arrowhead. This doublet could be an early degradation product or a difference in the translation start site of the mRNA. However, both bands decreased over time. The MAML1Δ75–301 protein also decreased over time but at a slower rate than MAML1 or MAML1-1-300. We also could not detect any degradation products with the MAML1Δ75–301 protein. ImageJ software was used to quantitate and normalize the different MAML1 protein levels. We normalized to both the over-expressed CBF1 and to native GAPDH because both proteins were stable over time. Fig 3B shows a graph summarizing the calculated half-lives of the different MAML proteins with standard deviations. The half-life of MAML1 and MAML1-1-301 were 155 and 170 minutes respectively. Interestingly, this is the very similar to the half-life previously reported for the NICD when MAML1 is overexpressed with it (7). The half-life of the MAML1Δ75–301 was determined to be 240 minutes and was statistically different than MAML1 using a student’s t-test (p = 0.0161) regardless of normalization to CBF1 or GAPDH. Taken together this suggested to us that the degron region of MAML1 was found in the 75–300 region of the protein.


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)

Half-life studies to MAML1-3 family members.(3A) MAML1, MAML1Δ75–301, MAML1-1-300, MAML2 or MAML3 were expressed with myc-tagged CBF1 in HeLa cells. Cells were treated with 150 μg/ml cycloheximide and cell extracts collected every hour for 5 hours. Western blots were performed to the myc-tag for the various proteins or to endogenously expressed GAPDH. Results shown are from representative blots form three different experiments. Black and gray arrowheads indicate degradation products that could be detected with time with the MAML1 and MAML1-300 constructs. (3B) ImageJ software was used to quantitate expression levels normalized to either CBF1 or GAPDH. The data is presented as the mean ± SD in bar graphs. A * indicates significantly different compared to MAML1 (p<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4520489&req=5

pone.0134013.g003: Half-life studies to MAML1-3 family members.(3A) MAML1, MAML1Δ75–301, MAML1-1-300, MAML2 or MAML3 were expressed with myc-tagged CBF1 in HeLa cells. Cells were treated with 150 μg/ml cycloheximide and cell extracts collected every hour for 5 hours. Western blots were performed to the myc-tag for the various proteins or to endogenously expressed GAPDH. Results shown are from representative blots form three different experiments. Black and gray arrowheads indicate degradation products that could be detected with time with the MAML1 and MAML1-300 constructs. (3B) ImageJ software was used to quantitate expression levels normalized to either CBF1 or GAPDH. The data is presented as the mean ± SD in bar graphs. A * indicates significantly different compared to MAML1 (p<0.05).
Mentions: We have also conducted pulse chase experiments to determine the half-life of MAML1, MAML1 1–300, and MAML1Δ75–301. Myc-tagged vectors for the different constructs were overexpressed with myc-tagged CBF1 in HeLa cells. Representative blots are shown in Fig 3A of the results of the pulse-chase studies conducted for the different MAML1 proteins. Over time, an increase in a degradation product was detected for MAML1. The black arrowhead shows a degradation product that is immediately detected at the first time point and increases over time with maximum amounts seen between 2 and 3 hours. The gray arrowhead shows a degradation product that is seen early, but only begins to increase in amount after the three hour time point. This same trend could also be seen for the MAML1-1-300. The MAML1-1-300 is detected as a doublet as indicated by the black arrowhead. This doublet could be an early degradation product or a difference in the translation start site of the mRNA. However, both bands decreased over time. The MAML1Δ75–301 protein also decreased over time but at a slower rate than MAML1 or MAML1-1-300. We also could not detect any degradation products with the MAML1Δ75–301 protein. ImageJ software was used to quantitate and normalize the different MAML1 protein levels. We normalized to both the over-expressed CBF1 and to native GAPDH because both proteins were stable over time. Fig 3B shows a graph summarizing the calculated half-lives of the different MAML proteins with standard deviations. The half-life of MAML1 and MAML1-1-301 were 155 and 170 minutes respectively. Interestingly, this is the very similar to the half-life previously reported for the NICD when MAML1 is overexpressed with it (7). The half-life of the MAML1Δ75–301 was determined to be 240 minutes and was statistically different than MAML1 using a student’s t-test (p = 0.0161) regardless of normalization to CBF1 or GAPDH. Taken together this suggested to us that the degron region of MAML1 was found in the 75–300 region of the protein.

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