Localization of MAML1 in HeLa cells.

<p>MAML1 was mutated at various lysine residues as described in text. Myc-tagged MAML1 Wild type (WT) or Mutant (KR) was expressed in HeLa cells. Twenty-four hours post-transfection, cells were either treated with DMSO or lactacystin (10 μM) and left for an additional 24 hours. Cells were then fixed in 4% paraformaldehyde and processed for immunocytochemistry. Both MAML1 and MAML1 KR mutant localize to nuclei in discrete foci. Images show MAML1 WT and KR with FITC stain alone, DAPI stain to detect nucleus along, or merged to show overlap. Scale Bar is 50 μm.</p

Half-life studies to MAML1-3 family members.

<p>(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).</p

Overview of Notch Signaling in the Nucleus.

<p>(A) In the absence of the NICD, Notch target genes remain in a repressed state through interaction of CBF1 with corepressor complexes (SMRT and HDAC1). (B) Release of the NICD from the cell membrane results in nuclear translocation and recruitment of MAML1, p300, and CDK8. CDK8 phosphorylates the NICD in the PEST domain as indicated by the lollipop structures. (C) Phosphorylation is thought to recruit the ubiquitin ligase Fbw7 to poly-ubiquitinate (boxes) the NICD, thereby signaling for degradation and shut off of target gene activation.</p

MAML1 ubiquitination is stimulated by p300 and inhibited by NICD.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134013#pone.0134013.g002" target="_blank">Fig 2</a> 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).</p

MAML1, CDK8 and p300 cooperatively stimulates Notch activity.

<p><b>(A)</b> HEK293 cells were co-transfected with a GAL4 luciferase gene and vectors expressing GAL4-Notch, MAML1, p300 and CDK8, as indicated in the figure. The data is presented as mean ± SD (n = 3). MAML1, p300, and CDK8 stimulate transcription in the presence of GAL4-Notch (*, p<0.0001). <b>(B)</b> Schematic of the <i>in vitro</i> transcription assay using chromatin templates. <b>(C)</b> Chromatin templates containing CSL binding sites were incubated with N1ICD, MAML1, CDK8, p300 and acetyl-CoA, as indicated.</p

MAML1 is Ubiquitinated.

<p>(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).</p

Identification of conserved lysine residues responsible for ubiquitination of MAML1.

<p>(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.</p

Proposed Model for Importance of MAML1 Ubiquitination.

<p>In the absence of activators (MEF2C, p53, NICD), MAML1 levels must remain low in the cell in order to prevent nonspecific transcriptional activation. MAML1 interacts with p300 to recruit an ubiquitin ligase to ubiquitinate MAML1 resulting in degradation. When activators are present, MAML1 is stabilized allowing co-transcriptional activation of target genes to occur.</p