Notch-induced E2A ubiquitination and degradation are controlled by MAP kinase activities - PubMed (original) (raw)

Notch-induced E2A ubiquitination and degradation are controlled by MAP kinase activities

Lei Nie et al. EMBO J. 2003.

Abstract

Notch signals are important for lymphocyte development but downstream events that follow Notch signaling are not well understood. Here, we report that signaling through Notch modulates the turnover of E2A proteins including E12 and E47, which are basic helix-loop-helix proteins crucial for B and T lymphocyte development. Notch-induced degradation requires phosphorylation of E47 by p42/p44 MAP kinases. Expression of the intracellular domain of Notch1 (N1-IC) enhances the association of E47 with the SCF(Skp2) E3 ubiquitin ligase and ubiquitination of E47, followed by proteasome-mediated degradation. Furthermore, N1-IC induces E2A degradation in B and T cells in the presence of activated MAP kinases. Activation of endogenous Notch receptors by treatment of splenocytes with anti-IgM or anti-CD3 plus anti-CD28 also leads to E2A degradation, which is blocked by the inhibitors of Notch activation or proteasome function. Notch-induced E2A degradation depends on the function of its downstream effector, RBP-Jkappa, probably to activate target genes involved in the ubiquitination of E2A proteins. Thus we propose that Notch regulates lymphocyte differentiation by controlling E2A protein turnover.

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Figures

Fig. 1. N1-IC inhibits E47 and E12 transcriptional activity by accelerating their degradation. (A) Dose responses. NIH3T3 cells were transiently transfected with 5 µg, 1 µg each of E-box luciferase and β-galactosidase reporter plasmids along with 1 µg of E47 or E12 plus increasing amounts of N1-IC expressing constructs. Luciferase activities were assayed 36 h later and normalized to those of β-galactosidase. Transcriptional activity of E47 in each sample is presented relative to that in the absence of N1-IC with standard deviations from at least three independent experiments. For protein analysis, indicated amounts of E47 or E12 and N1-IC were cotransfected with a GFP expression vector. Whole lysates were probed with anti-E47, GFP and N1-IC antibodies. (B) A proteasome inhibitor blocks the degradation of E47 protein induced by N1-IC. Immunoblot assays were carried out with NIH3T3 cells transfected with 1 µg of E47 expressing plasmid with or without 4 µg of the N1-IC construct and treated with the indicated amounts of MG-132 proteasome inhibitor, leupeptin or dimethylsulfoxide vehicle for 3 h prior to harvest. C indicates loading control. (C) Activation of endogenous Notch receptors enhances degradation of E47. The NIH3T3 fibroblasts expressing Notch ligand, Jagged-1 (J) or not (C), were infected with a retroviral construct producing both E47 and GFP. Cells were harvested 3 days after infection. Whole-cell lysates were analyzed using immunoblotting (IB) with antibodies against E47 and GFP. Nuclear extracts were used for detecting N1-IC. Another aliquot of the infected cells was used to isolate RNA for RT–PCR assays of HES1 and GAPDH mRNA.

Fig. 2. A functional domain mediating Notch1-induced inhibition. (A) Analysis of deletion mutants. In a schematic representation of E47 and mutants, activation and EHD domains are marked with patterned boxes and labeled. Sequences of the mutations in AD1 and AD2 are shown below the boxes with mutated residues underlined and followed by the substituting residues in parentheses. Expression of wild-type and mutant E47 in NIH3T3 cells was confirmed using immunoblots with antibodies against E47. One microgram of E47 or mutant-expressing plasmid was cotransfected with or without 2 µg of N1-IC-expressing constructs along with reporters into NIH3T3 cells and assayed as described for (Figure 1A). (B) Identification of MAP kinase sites in EHD3. Sequence of EHD3 is shown with MAP kinase sites labeled as M1–3. In the Mm and Sm constructs, serine or threonine shown in italic are substituted with alanine. NIH3T3 cells were cotransfected with 1 µg of E47 or mutant construct with reporter constructs and the indicated amounts of N1-IC construct. The reporter activities and protein levels were analyzed as described in Figure 1A.

Fig. 3. MAP kinase mediated phosphorylation of E47 is necessary but not sufficient for Notch1-induced degradation. (A) Phosphorylation of E47. Whole-cell lysates of NIH3T3 cells transfected with E47 or Mm with or without MEK1 were probed with anti-phospho-M2 peptide (SSPS

PVGSPQG) and then anti-E47 antibodies. Whole-cell lysates from Jurkat T and BaF3 B cells were analyzed similarly. (B) MAP kinases are necessary but not sufficient for Notch1-mediated degradation of E47 proteins. PD98059 or SB203850 were added at a final concentration of 50 µM for 2 h after transfection for 34 h. Protein levels were determined as described above. E47 levels normalized against loading controls are shown in the bar graph as relative levels to that in cells expressing E47 alone under each treatment. (C) MAP kinase activation by MEK1 and N1-IC. Transfected NIH3T3 cells were harvested 36 h post transfection and whole-cell lysates were used in immunoblotting with antibodies against phospho-p42/44 and p42/p44.

Fig. 4. N1-IC enhances ubiquitination of E47 protein in vivo by facilitating its association with the SCFSkp2 complex. (A) In vivo ubiquitination assay. Seven micrograms of HA-ubiquitin-expressing construct were transfected into 293T cells with 1 µg of E47 or Mm ± 3 µg of N1-IC or 1 µg of MEK1 together or individually as indicated. Transfected cells were treated 24 h later with MG132 at a final concentration of 4 µM overnight. Immunoprecipitation (IP) was performed with 800 µg of whole-cell lysates and anti-E47 antibodies. The precipitates were blotted with an anti-HA monoclonal antibody and then with anti-E47 antibodies. (B) Overexpression of E47 rescues p27 protein. Immunoblot assays were performed with 293T cells transfected with indicated constructs using antibodies against E47 and loading control. (C) Interaction between E47 and endogenous Skp2. Three micrograms of E47 (E) or Mm mutant (Mm) construct were cotransfected with or without 3 µg of MEK1 (M) or 9 µg of N1-IC (N) construct into 293T cells for 28 h as labeled at the top of each lane. Anti-Skp2 immunoprecipitates from whole-cell lysates were probed with antibodies against E47 and subsequently with antibodies against indicated proteins. (D) Interaction between E47 and exogenous Skp2. Co-IP assay was performed with 293T cells transfected with constructs indicated by their single-letter codes as described above except that 3 µg of the Skp2 (S) construct was included in all samples. An aliquot of total lysates was probed for pp42/44 and p42/44 in addition to E47 and Skp2. (E) Inhibition of E47 degradation by siRNA against Skp2. NIH3T3 cells were cotransfected with indicated constructs. Two days after transfection, whole-cell extracts were used for immunoblot assays of indicated proteins.

Fig. 5. MAP kinase activities are required for Notch1-induced E2A degradation. (A) The indicated cell lines or cultures were infected with N1-IC-expressing retrovirus (N) or vector control (V). Infected cells were sorted for GFP fluorescence. Endogenous E2A protein levels were detected using anti-E47 antibodies. The same membranes were reprobed with antibodies as indicated. The asterisk indicates the cleavage product of PARP. (B) Left: Levels of activated MAP kinases in cells used in (A). Right: kinetics of MAP kinase activation in total splenocytes treated with Con A and LPS. Whole-cell lysates were analyzed. Whole-cell lysates from vector or N1-IC-infected 16610D (C) or BaF3 (D) cells were analyzed with the indicated antibodies with or without treatment with 10 ng/ml PMA for 2 and 4 h (C) or 50 µM PD98049 for 3 h (D). (E) Interaction between endogenous E2A and Skp2. Co-IP was performed with antibodies against Skp2 or a preimmune serum and whole-cell lysates from the indicated cells. The precipitates were probed for E2A and Skp2.

Fig. 6. E2A degradation by endogenous Notch signals. Activation of Notch-signaling pathways and E2A degradation in splenic cultures treated with antibodies against (A) IgM or (B) CD3 and CD28. Total RNA was isolated from splenocytes cultured under the indicated conditions for 2 or 3 h. Expression of HES1 and β-actin was measured using RT–PCR. Whole-cell lysates from splenocytes cultured under the same conditions were used for immunoblot assays with antibodies against the indicated proteins. Z-IL-CHO or an equal volume of the vehicle was added to the indicated cultures. (C) Proteasome inhibitor blocks E2A degradation. MG132 was included in the 2 h culture as described in (B). (D) Ubiquitination of E2A proteins in a 3 h culture of splenocytes treated with 25 µM MG132 plus a mixture of antibodies against CD3, CD28 and IgM as described in Materials and methods; 108 cells were used for each IP with a preimmune serum (pre) or anti-E47 (E47) antibodies. Immunoblots of the precipitates were performed with the indicated antibodies.

Fig. 7. Notch signals leading to E2A degradation. (A) Analyses of N1-IC deletion mutants and Deltex. NIH 3T3 cells were transfected with 1 µg of E47 and 4 µg of N1-IC or Deltex constructs. Protein levels were analyzed 2 days later as described above. Wild-type and mutant N1-IC proteins are indicated with arrowheads. (B) Jagged-1-expressing NIH3T3 cells were transfected with indicated constructs. Protein (left) and luciferase and β-galactosidase activities (right) were measured as described. (C) NIH3T3 cells were transfected with the indicated constructs and protein levels were analyzed using immunoblots. (D) A model for Notch-induced E2A degradation. E2A proteins are phosphorylated by MAP kinases, which allows association with the SCFskp2 E3 ubiquitin ligase. The ubiquitinated E2A proteins are degraded by the 26S proteasome. Notch signals enhance the ubiquitination of E2A proteins, probably by activating expression of their downstream target genes involved in the ubiquitination reaction. Notch signals also activate MAP kinases in some cell types.

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Notch-induced E2A ubiquitination and degradation are controlled by MAP kinase activities - PubMed (original) (raw)