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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

Identification of conserved lysine residues responsible for ubiquitination of MAML1.(A) MAML1 was mutated at various lysine residues as described in text. Myc-tagged MAML1 Wild type (WT) or Mutant (K/R) was expressed with HA-tagged ubiquitin and immunoprecipitated to the myc tag. Western blots were performed to HA (upper blot) or myc (lower blot) for loading control. Ubiquitination was decreased by greater than 95% in the mutant compared to wild type MAML1. (B) MAML1 K/R mutant activates a HES1 reporter construct stronger than MAML1 WT. HeLa cells were co-transfected with HES1-luciferase reporter construct, renilla luciferase, N1ICD, and either MAML1 WT or MAML1K/R. HES1-Luciferase activity assays were performed and results shown are the mean ± SD (n = 4). *, p<0.05.
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pone.0134013.g004: Identification of conserved lysine residues responsible for ubiquitination of MAML1.(A) MAML1 was mutated at various lysine residues as described in text. Myc-tagged MAML1 Wild type (WT) or Mutant (K/R) was expressed with HA-tagged ubiquitin and immunoprecipitated to the myc tag. Western blots were performed to HA (upper blot) or myc (lower blot) for loading control. Ubiquitination was decreased by greater than 95% in the mutant compared to wild type MAML1. (B) MAML1 K/R mutant activates a HES1 reporter construct stronger than MAML1 WT. HeLa cells were co-transfected with HES1-luciferase reporter construct, renilla luciferase, N1ICD, and either MAML1 WT or MAML1K/R. HES1-Luciferase activity assays were performed and results shown are the mean ± SD (n = 4). *, p<0.05.

Mentions: MAML1 contains three well characterized domains (Fig 2A): the N-terminal acidic domain which contains the first 75 amino acids and binds to NICD, the basic domain (75–301aa) which binds to p300 and the c-terminal acidic domain which has not been shown to interact with a protein. Our results suggested that ubiquitination might be occurring around the 75–300 region because deletion of this region resulted in an increase in half-life and ubiquitination could not be detected. Therefore, we began mutating a series of lysine residues in MAML1 and testing for a decrease in ubiquitination. As shown in Fig 4A, in order to remove >95% of the ubiquitination from the over-expressed protein, we needed to mutate 8 lysine residues to arginine (K112, 178, 188, 189, 405, 407, 639, 822R) and have designated this construct as MAML1K/R for simplicity. Both the MAML1 and MAML1K/R proteins were over-expressed at the same level indicating that the decrease in ubiquitination was due to mutation of K residues to R residues and not overall change in recombinant protein levels.


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)

Identification of conserved lysine residues responsible for ubiquitination of MAML1.(A) MAML1 was mutated at various lysine residues as described in text. Myc-tagged MAML1 Wild type (WT) or Mutant (K/R) was expressed with HA-tagged ubiquitin and immunoprecipitated to the myc tag. Western blots were performed to HA (upper blot) or myc (lower blot) for loading control. Ubiquitination was decreased by greater than 95% in the mutant compared to wild type MAML1. (B) MAML1 K/R mutant activates a HES1 reporter construct stronger than MAML1 WT. HeLa cells were co-transfected with HES1-luciferase reporter construct, renilla luciferase, N1ICD, and either MAML1 WT or MAML1K/R. HES1-Luciferase activity assays were performed and results shown are the mean ± SD (n = 4). *, p<0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520489&req=5

pone.0134013.g004: Identification of conserved lysine residues responsible for ubiquitination of MAML1.(A) MAML1 was mutated at various lysine residues as described in text. Myc-tagged MAML1 Wild type (WT) or Mutant (K/R) was expressed with HA-tagged ubiquitin and immunoprecipitated to the myc tag. Western blots were performed to HA (upper blot) or myc (lower blot) for loading control. Ubiquitination was decreased by greater than 95% in the mutant compared to wild type MAML1. (B) MAML1 K/R mutant activates a HES1 reporter construct stronger than MAML1 WT. HeLa cells were co-transfected with HES1-luciferase reporter construct, renilla luciferase, N1ICD, and either MAML1 WT or MAML1K/R. HES1-Luciferase activity assays were performed and results shown are the mean ± SD (n = 4). *, p<0.05.
Mentions: MAML1 contains three well characterized domains (Fig 2A): the N-terminal acidic domain which contains the first 75 amino acids and binds to NICD, the basic domain (75–301aa) which binds to p300 and the c-terminal acidic domain which has not been shown to interact with a protein. Our results suggested that ubiquitination might be occurring around the 75–300 region because deletion of this region resulted in an increase in half-life and ubiquitination could not be detected. Therefore, we began mutating a series of lysine residues in MAML1 and testing for a decrease in ubiquitination. As shown in Fig 4A, in order to remove >95% of the ubiquitination from the over-expressed protein, we needed to mutate 8 lysine residues to arginine (K112, 178, 188, 189, 405, 407, 639, 822R) and have designated this construct as MAML1K/R for simplicity. Both the MAML1 and MAML1K/R proteins were over-expressed at the same level indicating that the decrease in ubiquitination was due to mutation of K residues to R residues and not overall change in recombinant protein levels.

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