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. 2009 Nov;37(20):6691-700.
doi: 10.1093/nar/gkp724. Epub 2009 Sep 8.

GSK3beta is a negative regulator of the transcriptional coactivator MAML1

Mariana Saint Just Ribeiro  1 , Magnus L Hansson, Mikael J Lindberg, Anita E Popko-Scibor, Annika E Wallberg
Affiliations

GSK3beta is a negative regulator of the transcriptional coactivator MAML1

Mariana Saint Just Ribeiro et al. Nucleic Acids Res. 2009 Nov.

Abstract

Glycogen synthase kinase 3beta (GSK3beta) is involved in several cellular signaling systems through regulation of the activity of diverse transcription factors such as Notch, p53 and beta-catenin. Mastermind-like 1 (MAML1) was originally identified as a Notch coactivator, but has also been reported to function as a transcriptional coregulator of p53, beta-catenin and MEF2C. In this report, we show that active GSK3beta directly interacts with the MAML1 N-terminus and decreases MAML1 transcriptional activity, suggesting that GSK3beta might target a coactivator in its regulation of gene expression. We have previously shown that MAML1 increases global acetylation of histones, and here we show that the GSK3 inhibitor SB41, further enhances MAML1-dependent histone acetylation in cells. Finally, MAML1 translocates GSK3beta to nuclear bodies; this function requires full-length MAML1 protein.

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Figures

Figure 1.
Figure 1.
GSK3β decreases MAML1 activity. (A) HEK-293 cells were cotransfected with a luciferase reporter containing five GAL4 binding sites and vectors expressing GAL4-MAML1 and GSK3β as indicated. (B) GAL4-MAML1 was cotransfected into HEK-293 cells with the luciferase reporter in the absence or presence of SB41. (C) HEK-293 cells were transfected with a vector expressing FLAG-MAML1, MAML1 was affinity-purified with M2-agarose, and proteins separated by SDS–PAGE. Immunoblot detected MAML1, and GSK3β protein that interacted with MAML1. (D) Endogenous MAML1 protein was immunoprecipitated from C33A cells with an antibody recognizing the N-terminus of MAML1. Proteins were separated by SDS–PAGE and detection of MAML1, and MAML1-interacting proteins, was monitored by immunoblot with antibodies recognizing MAML1 and GSK3β. (E) GST-MAML1 and GST coupled to glutathione-Sepharose beads were incubated with affinity-purified HIS-GSK3β, and MAML1-interacting GSK3β was monitored by immunoblot. The input represents 10% of GSK3β used in the binding reaction. (F) GST-GSK3β and GST coupled to glutathione-Sepharose beads were incubated with affinity-purified FLAG-MAML1, and MAML1 binding to GSK3β was monitored by immunoblot. The input represents 10% of the MAML1 used in the binding reaction. (G) GSK3β phosphorylates MAML1 in vitro. Recombinant affinity-purified proteins HIS-GSK3β, GST-MAML1 or GST, were incubated with [γ-32P]ATP, in the presence or absence of SB41, proteins were separated by SDS–PAGE and visualized by autoradiography.
Figure 2.
Figure 2.
Active GSK3β regulates MAML1 transcription. (A) Vectors expressing GAL4-MAML1 and HA-GSK3β as WT, S9A or K85R, were cotransfected into HEK-293 cells with a luciferase reporter. (B) Vectors expressing MAML1 and GSK3β were cotransfected into HEK-293 cells as indicated, whole-cell extracts were prepared, and MAML1 protein immunoprecipitated. Proteins were separated by SDS–PAGE and detection of MAML1, and MAML1-interacting proteins, was monitored by immunoblot with antibodies recognizing MAML1 and GSK3β.
Figure 3.
Figure 3.
GSK3β directly interacts with the MAML1 N-terminus. (A) Schematic diagram of the MAML1 protein. GST-tagged MAML1 proteins as indicated, were incubated with whole-cell extract from HEK-293 cells transfected with pCDNA-GSK3β (B) or recombinant affinity-purified GSK3β (C), and MAML1-interacting GSK3β detected with immunoblot. (D) Recombinant HIS-GSK3β and GST-MAML1 proteins were affinity-purified and incubated with [γ-32P]ATP. Proteins were separated by SDS–PAGE and visualized by autoradiography. (E) GST-MAML1 proteins were incubated with HIS-GSK3β in the presence of cold ATP. The kinase reactions were separated by SDS–PAGE and stained with Pro-Q Diamond phosphoprotein staining kit. (F) Vectors expressing GSK3β and were cotransfected with a luciferase reporter containing five GAL4 binding sites into U2OS cells.
Figure 4.
Figure 4.
MAML1 directs GSK3β to nuclear bodies. (A) Vectors expressing FLAG-MAML1 domains as indicated, and HA-GSK3β were cotransfected into Cos7 cells, and after 24 h the cells were immunostained with antibodies recognizing the FLAG- and HA-tags. (B) Vectors expressing HA-GSK3β-S9A, HA-GSK3β-K85R and FLAG-MAML1 were cotransfected into Cos7 cells, and cells were immunostained after 24 h with antibodies recognizing the FLAG- and HA-tags. (C) Cos7 cells were transfected with FLAG-MAML1 (or empty vector) and immunostained after 24 h with antibodies recognizing the FLAG-tag, and endogenous GSK3β phosphorylated at serine 9 (P-S9).
Figure 5.
Figure 5.
The MAML1-Notch1 ICD interaction is not abolished by GSK3β. (A) Vectors expressing FLAG-Notch1 ICD, HA-GSK3β-S9A and MAML1 1–1016 were cotransfected into Cos7 cells, and after 24 h cells were immunostained with antibodies recognizing the FLAG- or HA-tags or MAML1. (B) HEK-293 cells were cotransfected with a luciferase reporter containing five GAL4 binding sites and vectors expressing GAL4-Notch1 ICD, MAML1 and GSK3β. (C) C33A cells were transfected with Notch1 ICD and siRNA MAML1 or control (ctrl) siRNA, and incubated in the presence or absence of SB41. Expression of MAML1, GAPDH and the Notch target gene Hes1 was analyzed with immunoblot with antibodies recognizing MAML1, Hes1 and GAPDH. (D) GST-MAML1 and GST were incubated, in the presence or absence of ATP, with affinity-purified HIS-GSK3β and FLAG-Notch1 ICD proteins as indicated, and MAML1-interacting proteins were detected with immunoblot.
Figure 6.
Figure 6.
GSK3β inhibits MAML1-dependent global histone acetylation. f-MAML1 or HEK-293 cells (ctrl cell line), were incubated in the presence or absence of SB41. The levels of expressed MAML1, acetylated histone H3 (Ac-H3), and tubulin, were monitored with immunoblot.
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