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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 is Ubiquitinated.(A) Cartoon depiction of MAML1 and the deletion constructs used in this study. MAML1 has been shown to have three domains, two acidic domains as indicated by red and one basic domain as indicated by blue. The acidic domain at the N-terminus (1–75 aa) has been shown to interact with the N1ICD. The central basic domain (75–300 aa) has been shown to interact with p300. The c-terminus domain has not been shown to interact with any proteins. (B) HES1 is activated by a combination of N1ICD with MAML1. Deletion of various domains of MAML1 show decreased reporter activity. Results are normalized to renilla luciferase and fold activation is compared to HES1 alone (n = 3). (C) MAML1 is ubiquitinated. MAML1 was overexpressed with HA-Ub. MAML1 was immunoprecipitated and western blots were performed to HA (Top blot) or MAML1 (lower blot) to show expression levels. (D) Ubiquitination of MAML1 occurs in the first 300 amino acids. MAML1 full-length, MAML1-1-300 or MAML1-300-1016 were expressed with HA-Ub and immunoprecipitated. Western blots were performed to ubiquitin (HA, top blot) or MAML1 constructs (Myc, bottom blot).
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pone.0134013.g002: MAML1 is Ubiquitinated.(A) Cartoon depiction of MAML1 and the deletion constructs used in this study. MAML1 has been shown to have three domains, two acidic domains as indicated by red and one basic domain as indicated by blue. The acidic domain at the N-terminus (1–75 aa) has been shown to interact with the N1ICD. The central basic domain (75–300 aa) has been shown to interact with p300. The c-terminus domain has not been shown to interact with any proteins. (B) HES1 is activated by a combination of N1ICD with MAML1. Deletion of various domains of MAML1 show decreased reporter activity. Results are normalized to renilla luciferase and fold activation is compared to HES1 alone (n = 3). (C) MAML1 is ubiquitinated. MAML1 was overexpressed with HA-Ub. MAML1 was immunoprecipitated and western blots were performed to HA (Top blot) or MAML1 (lower blot) to show expression levels. (D) Ubiquitination of MAML1 occurs in the first 300 amino acids. MAML1 full-length, MAML1-1-300 or MAML1-300-1016 were expressed with HA-Ub and immunoprecipitated. Western blots were performed to ubiquitin (HA, top blot) or MAML1 constructs (Myc, bottom blot).

Mentions: MAML1 has been previously shown to be a general co-activator of numerous transcription factors including MEF2C, p53, and Notch. Overexpression of MAML1 with N1ICD increased HES1 promoter activation compared to HES1 or HES1+N1ICD alone which is in agreement with previous reports (Fig 2B). MAML1 1–710, MAML1Δ75–301 and MAML1 1–301 (Fig 2A) had reduced activation of the HES1 promoter construct compared to MAML1. Further, MAML1 1–301 has also been shown to act as a dominant negative in vivo [7]. Our results suggested to us that either MAML1 could recruit other unknown factors that interact with the C-terminus or that the C-terminus could be modified. Previous reports have shown MAML1 to be phosphorylated [23] and acetylated [25]. Since phosphorylation has been shown to be required for ubiquitination of other transcription factors, we determined if MAML1 could be ubiquitinated. In Fig 2C, we show that the full-length MAML1 protein can be ubiquitinated. The ubiquitination was specific to MAML1 because overexpression of either Myc-MAML1 or HA-ubiquitin alone followed by IP to myc and western blot to HA did not show any detectable bands (Fig 2C, lanes 1 and 3).


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 is Ubiquitinated.(A) Cartoon depiction of MAML1 and the deletion constructs used in this study. MAML1 has been shown to have three domains, two acidic domains as indicated by red and one basic domain as indicated by blue. The acidic domain at the N-terminus (1–75 aa) has been shown to interact with the N1ICD. The central basic domain (75–300 aa) has been shown to interact with p300. The c-terminus domain has not been shown to interact with any proteins. (B) HES1 is activated by a combination of N1ICD with MAML1. Deletion of various domains of MAML1 show decreased reporter activity. Results are normalized to renilla luciferase and fold activation is compared to HES1 alone (n = 3). (C) MAML1 is ubiquitinated. MAML1 was overexpressed with HA-Ub. MAML1 was immunoprecipitated and western blots were performed to HA (Top blot) or MAML1 (lower blot) to show expression levels. (D) Ubiquitination of MAML1 occurs in the first 300 amino acids. MAML1 full-length, MAML1-1-300 or MAML1-300-1016 were expressed with HA-Ub and immunoprecipitated. Western blots were performed to ubiquitin (HA, top blot) or MAML1 constructs (Myc, bottom blot).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134013.g002: MAML1 is Ubiquitinated.(A) Cartoon depiction of MAML1 and the deletion constructs used in this study. MAML1 has been shown to have three domains, two acidic domains as indicated by red and one basic domain as indicated by blue. The acidic domain at the N-terminus (1–75 aa) has been shown to interact with the N1ICD. The central basic domain (75–300 aa) has been shown to interact with p300. The c-terminus domain has not been shown to interact with any proteins. (B) HES1 is activated by a combination of N1ICD with MAML1. Deletion of various domains of MAML1 show decreased reporter activity. Results are normalized to renilla luciferase and fold activation is compared to HES1 alone (n = 3). (C) MAML1 is ubiquitinated. MAML1 was overexpressed with HA-Ub. MAML1 was immunoprecipitated and western blots were performed to HA (Top blot) or MAML1 (lower blot) to show expression levels. (D) Ubiquitination of MAML1 occurs in the first 300 amino acids. MAML1 full-length, MAML1-1-300 or MAML1-300-1016 were expressed with HA-Ub and immunoprecipitated. Western blots were performed to ubiquitin (HA, top blot) or MAML1 constructs (Myc, bottom blot).
Mentions: MAML1 has been previously shown to be a general co-activator of numerous transcription factors including MEF2C, p53, and Notch. Overexpression of MAML1 with N1ICD increased HES1 promoter activation compared to HES1 or HES1+N1ICD alone which is in agreement with previous reports (Fig 2B). MAML1 1–710, MAML1Δ75–301 and MAML1 1–301 (Fig 2A) had reduced activation of the HES1 promoter construct compared to MAML1. Further, MAML1 1–301 has also been shown to act as a dominant negative in vivo [7]. Our results suggested to us that either MAML1 could recruit other unknown factors that interact with the C-terminus or that the C-terminus could be modified. Previous reports have shown MAML1 to be phosphorylated [23] and acetylated [25]. Since phosphorylation has been shown to be required for ubiquitination of other transcription factors, we determined if MAML1 could be ubiquitinated. In Fig 2C, we show that the full-length MAML1 protein can be ubiquitinated. The ubiquitination was specific to MAML1 because overexpression of either Myc-MAML1 or HA-ubiquitin alone followed by IP to myc and western blot to HA did not show any detectable bands (Fig 2C, lanes 1 and 3).

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