Coordinated regulation of transcription factor Bcl11b activity in thymocytes by the mitogen-activated protein kinase (MAPK) pathways and protein sumoylation

Abstract

The transcriptional regulatory protein Bcl11b is essential for T-cell development. We have discovered a dynamic, MAPK-regulated pathway involving sequential, linked, and reversible post-translational modifications of Bcl11b in thymocytes. MAPK-mediated phosphorylation of Bcl11b was coupled to its rapid desumoylation, which was followed by a subsequent cycle of dephosphorylation and resumoylation. Additionally and notably, we report the first instance of direct identification by mass spectrometry of a site of small ubiquitin-like modifier (SUMO) adduction, Lys-679 of Bcl11b, in a protein isolated from a native, mammalian cell. Sumoylation of Bcl11b resulted in recruitment of the transcriptional co-activator p300 to a Bcl11b-repressed promoter with subsequent induction of transcription. Prolonged treatment of native thymocytes with phorbol 12,13-dibutyrate together with the calcium ionophore A23187 also promoted ubiquitination and proteasomal degradation of Bcl11b, providing a mechanism for signal termination. A Bcl11b phospho-deSUMO switch was identified, the basis of which was phosphorylation-dependent recruitment of the SUMO hydrolase SENP1 to phospho-Bcl11b, coupled to hydrolysis of SUMO-Bcl11b. These results define a regulatory pathway in thymocytes that includes the MAPK pathways and upstream signaling components, Bcl11b and the associated nucleosome remodeling and deacetylation (NuRD) complex, SENP proteins, the Bcl11b protein phosphatase 6, the sumoylation machinery, the histone acetyltransferase p300, and downstream transcriptional machinery. This pathway appears to facilitate derepression of repressed Bcl11b target genes as immature thymocytes initiate differentiation programs, biochemically linking MAPK signaling with the latter stages of T-cell development.

FIGURE 1.

Regulated sumoylation of Bcl11b in primary thymocytes. A, time course of the appearance of slowly migrating Bcl11b species (asterisks) in stimulated thymocytes. Primary thymocytes were treated with P/A as indicated, and cells were lysed in denaturing sample buffer prior to electrophoresis and immunoblotting analyses using antibodies for Bcl11b, phospho-Erk1/2 (p-Erk), phospho-p38 kinase (p-p38), and HDAC1 indicated at right. The HDAC1 blot serves as the loading control. The filled arrowhead in the top panel depicts a P/A-induced shift in the electrophoretic mobility of Bcl11b, and the open arrowhead indicates when that electrophoretic mobility shift is reversed. B and C, P/A-induced sumoylation of Bcl11b in thymocytes. Thymocytes were treated with P/A for the indicated times prior to lysis, immunoprecipitated with the anti-SUMO antibodies indicated, and immunoblotted using an anti-Bcl11b antibody. These representative findings have been replicated multiple times using several experimental approaches. Note that mouse thymocytes express two splice variants of Bcl11b; the shorter form is composed of exons 1, 2, and 4 and migrates just under the 130-kDa marker, and a longer form is composed of exons 1, 2, 3, and 4 and is 72 amino acids longer than the short form and migrates at the 130-kDa marker.

FIGURE 2.

Mapping Bcl11b sumoylation sites. Identification of Bcl11b Lys-679 as a site of SUMO1 (A) and SUMO2/3 (B) adduction in primary thymocytes is shown. LTQ ion trap tandem mass spectra are shown with FT-ICR full-scan mass spectra (insets). The derived experimental mass was 2751.3200 Da (error from theoretical, 1.4 ppm) for the SUMO1 adduct and 4468.1021 Da (error from theoretical, 0.11 ppm) for the SUMO2/3 adduct. C, identification of Lys-679 and Lys-877 as Bcl11b sumoylation sites by site-directed mutagenesis. HEK293T cells were co-transfected with expression vectors encoding Bcl11b or the mutants indicated (1 μg) and HA-SUMO1 or HA-SUMO2 (2 μg). The indicated SUMO adducts were deduced from sumoylation patterns of single point mutants where “n” indicates the likely presence of SUMO chains. The results shown in C have been replicated three to five times. Note that the experiment depicted in C was conducted using the form of Bcl11b corresponding to exons 1, 2, and 4, and consequently, the SUMO adducts of this splice variant are somewhat smaller than those of native thymocytes (which express splice variants composed of exons 1, 2, and 4 and exons 1, 2, 3, and 4; see Fig. 1).

FIGURE 3.

Phosphorylation of Bcl11b by MAPK pathways. A, time course of Bcl11b phosphorylation. Immunoprecipitation experiments were carried out using extracts from thymocytes treated with P/A as indicated. The upper blot was probed with an anti-phospho-Thr/Ser antibody (anti-pT/S). The blot was stripped and reprobed with an anti-Bcl11b antibody (lower blot). B, activated Erk and p38 kinases contributed to Bcl11b phosphorylation at 5 min. Thymocytes were preincubated with the MEK inhibitor U0126 (U0; 10 μm) or the p38 inhibitor SB202190 (SB; 10 μm) before treating with P/A for 5 min. Samples were immunoprecipitated, and immunoblots were generated as in A. C, inhibition of the Erk pathway, but not that of p38, blocks P/A-induced Bcl11b dephosphorylation at 30 min. Thymocytes were preincubated with U0126 or SB202190 as indicated before treating with P/A for 60 min. Samples were processed as in A. D, the PP1/PP2A phosphatase inhibitor calyculin A (Cal A) stimulated phosphorylation and blocked sumoylation of Bcl11b. Thymocytes were treated with 50 nm calyculin A for the indicated times, and samples were processed as in A.

FIGURE 4.

Chromatographic resolution of isobaric phosphosite isomers of Bcl11b monophosphopeptide 310–322. A, top, FT-ICR extracted ion current (XIC) chromatogram and mass determinations of the leading and trailing peaks are presented compared with a theoretical m/z of 728.37343. Vertical lines indicate the time at which tandem mass spectra were recorded. Bottom, LTQ ion trap tandem mass spectra recorded at 26.79 min (positive ordinate) and 28.05 min (negative ordinate) elution time identify the leading peak in the top panel as Thr(P)-313 and the trailing peak as Ser(P)-318 localized to monophosphopeptide 310–322. See supplemental Fig. S1 for additional spectra identifying Bcl11b phosphorylation sites in native thymocytes. B, schematic representation of Bcl11b with 23 phosphorylation and two sumoylation sites. ZnF indicates zinc finger domains. RT, retention time. th, mass-to-charge ratio in thomsons.

FIGURE 5.

SENP1 preferentially interacts with phosphorylated Bcl11b. Immunoprecipitation and immunoblotting were conducted as indicated in all panels. A, desumoylation of HA-SU1-Bcl11b and HA-SU2-Bcl11b in transfected HEK cells by both SENP1 and SENP2. The Bcl11b blot (lower image) was generated by stripping and reprobing the upper blot. B, interaction of SENP1 with Bcl11b complexes in transfected HEK293T cells. C, sumoylation is not required for the interaction between Bcl11b and SENP1. HEK293 cells were transfected with an expression vectors encoding wild-type Bcl11b or the double mutant 2R. Whole cell extracts from transfected HEK cells were immunoprecipitated with an anti-Bcl11b antibody, and the immunoprecipitates were probed with anti-Bcl11b (upper blot) or anti-FLAG (SENP1; lower blot) antibody. D, dephosphorylation of Bcl11b by HA-PP6 in transiently transfected HEK 293T cells; asterisks represent nonspecific immunoreactivity in input. E, dephosphorylation of Bcl11b inhibits interaction of SENP1 with the Bcl11b complex. The amount of expression vectors transfected in A–E were as follows: Bcl11b, 1 μg; HA-SUMO1 or -SUMO2, 2 μg; HA-SENP1 or -SENP2, 2 μg; HA-PP6, 2 μg where indicated. F, Bcl11b phosphorylation/sumoylation time course. Primary thymocytes were treated with P/A for 5 min to achieve maximum Bcl11b phosphorylation or 60 min to induce dephosphorylation and sumoylation of Bcl11b as indicated. Bcl11b was immunoprecipitated, and the immunoprecipitates were probed with anti-phospho-Thr/Ser (anti-pT/S) (upper blot) or anti-SUMO1 (middle blot) antibody, and the blot was then stripped and probed with an anti-Bcl11b antibody (lower blot). G, native SENP1 preferentially interacts with phosphorylated Bcl11b in primary thymocytes. Primary thymocytes were treated with P/A for 5 min to achieve maximum Bcl11b phosphorylation or 60 min to induce dephosphorylation of Bcl11b as indicated. H, mutation of Bcl11b phosphorylation sites decreases the affinity of SENP1 for the Bcl11b complex in transfected HEK cells. HEK293T cells were co-transfected with expression vectors encoding FLAG-tagged SENP1 (F-SENP1) and increasing amounts of FLAG-tagged WT or phosphomutant Bcl11b pMT as indicated. Protein levels of SENP1 and Bcl11b were quantified as described under “Experimental Procedures.” The graph reflects relative protein levels of immunoprecipitated Bcl11b (x axis) and co-immunoprecipitated SENP1 (y axis) at each level of Bcl11b. Asymptotic levels of WT Bcl11b and co-immunoprecipitated SENP1 were arbitrarily set as 100%. Each data point and error bar represents the mean ± S.E., respectively, of three independent determinations. The curves shown were derived from fitting data using a non-linear routine and parameters described in Equation 1. The fitted K values from the theoretical curves are 49.6 ± 6.4 and 84.2 ± 9.8 arbitrary units for wild-type Bcl11b and Bcl11b pMT, respectively, and the difference between these parameter estimates is statistically significant (p < 0.05 level; n = 3).

FIGURE 6.

Effect of sumoylation site mutations and phosphorylation on Bcl11b-mediated transcriptional repressive activity. A, diagram of CAT reporter construct harboring the mouse Id2 promoter. An ∼3-kb fragment of the mouse Id2 promoter from −2919 bp upstream to +152 bp downstream of the TSS was cloned to a promoterless CAT reporter plasmid. Boxes represent putative binding sites for the following transcription factors: E box factors (E), RFX1 (R), GATA1 (G), and CdxA (C). B, expression vectors encoding wild-type Bcl11b, the sumoylation-deficient mutants K679R and K877R, or the double mutant K679R/K877R (400 ng of each) were co-transfected into HEK293T cells with the Id2-CAT reporter (see A) as indicated. Relative CAT expression was measured as described previously (53). Note that this experiment was conducted in the absence of a co-transfected expression vector encoding a SUMO protein. C, time course of PDBu-induced phosphorylation of Bcl11b in transfected HEK293T cells. Cultured cells were serum-starved overnight prior to addition of serum with and without PDBu (100 nm) for the indicated times. Note that addition of serum without PDBu also induced Bcl11b phosphorylation (odd-numbered lanes) that differed from PDBu-induced phosphorylation (even-numbered lanes) in the extent of Bcl11b phosphorylation. D, sensitivity of PDBu (100 nm)-induced phosphorylation of Bcl11b to inhibition by U0126 (U0; 10 μm) and SB202190 (SB; 10 μm). Cells were transfected with an expression vector encoding Bcl11b (1 μg) and treated with PDBu with or without the indicated inhibitors for 4 h. E, effect of PDBu treatment with and without inhibitors U0126 and SB202190 on Bcl11b-mediated repression of the Id2 promoter. Cells were transfected with a Bcl11b expression vector or pcDNA3 (400 ng each). All treatments were conducted for 24 h prior to harvesting and processing for reporter gene assays as described (53). Bars and error bars in panels B and E represent the average ± S.E., respectively, of at least three independent experiments. Statistical significance between the indicated pairs was determined using the Student's t test (*, p ≤ 0.05; **, p ≤ 0.01; n > 3 in all cases). anti-pT/S, anti-phospho-Thr/Ser.

FIGURE 7.

Sumoylated Bcl11b interacts with the transcriptional co-activator p300. A, time course of Id2 induction in thymocytes by P/A treatment. Primary thymocytes were stimulated with P/A for the times indicated prior to RT-qPCR analyses of Id2 expression. Fold change in Id2 mRNA levels was calculated relative to the expression of a housekeeping gene, Gapdh. B, U0126 (U0), but not SB202190 (SB), inhibited the induction of the Id2 promoter by P/A treatment. C, effect of P/A treatment on interaction of Bcl11b and cofactors with Id2 promoter. Thymocytes were stimulated with P/A for the indicated times, and cells were subjected to ChIP analyses using the indicated antibodies or IgG. Chromatin was analyzed by qPCR using primers covering the −3-kb region of the Id2 promoter (left) that harbors a Bcl11b binding site, the TSS (middle), or 3′-intergenic region (IGR) (right). Points represent the relative ratio to input for each ChIP. D and E, P/A treatment promotes interaction of p300 with the Bcl11b complex. Primary thymocytes were treated with P/A for 60 min followed by reciprocal immunoprecipitations and immunoblotting as indicated. F, mutation of Bcl11b sumoylation sites inhibits interaction of p300 with the Bcl11b complex. HEK293T cells were transfected with 1 μg of expression vectors encoding Bcl11b (WT) or the double SUMO site deletion mutant of Bcl11b (2R) as indicated. Immunoprecipitation and immunoblotting were conducted as indicated. G, Bcl11b and p300 co-occupy Id2 promoter in thymocytes after P/A treatment. Primary thymocytes were stimulated with P/A for 60 min prior to re-ChIP analyses using anti-Bcl11b (first antibody) and anti-p300 or control IgG (second antibody). Immunoprecipitated DNA was analyzed by qPCR as indicated in C. Bars and error bars in panels A, B, and G represent the average ± S.E., respectively, of at least three independent experiments. H, induction of the Id2 promoter by P/A requires Bcl11b. P/A treatment of thymocytes was conducted, and Id2 transcripts were analyzed by RT-qPCR with relative quantification. All treatment values for each mouse are expressed relative to basal Id2 expression in thymocytes from the same mouse. Basal expression of Id2 was arbitrarily set to unity in all cases. The thymocytes used were from mice in which both alleles of the Bcl11b locus was floxed (Bcl11b^L2/L2) or excised at the DP stage (Bcl11b^dp−/−) (24). Bars and error bars in panel H represent the level of Id2 transcripts and S.E., respectively, in individual mice of the indicated genotype. Statistical significance throughout the figure was determined using Student's t test (*, p < 0.05; ***, p < 0.001). CalA, calyculin A.

FIGURE 8.

Fusion to SUMO1 and SUMO2 abrogates Bcl11b-mediated repression. A, fusion to SUMO1 abrogates Bcl11b-mediated repression. HEK293T cells were co-transfected with the Id2-CAT reporter construct and 200, 400, or 800 ng of expression vectors encoding wild-type Bcl11b or the HA-SUMO1-Bcl11b (HA-SU1-Bcl11b) fusion construct as indicated. Similar levels of Bcl11b and SUMO1-Bcl11b protein were expressed as shown in the immunoblot below the figure. B, immunocytochemical nuclear/subnuclear distribution of Bcl11b and SU1-Bcl11b. Fusion to SUMO1 did not influence expression levels or the nuclear/subnuclear distribution of Bcl11b. HEK293T cells were transfected with either Bcl11b or SU1-Bcl11b as indicated, and then cells were fixed and subjected to immunocytochemistry analysis using anti-Bcl11b antibody (green). The nuclei were counterstained with DAPI. The white size bar in the lower left panel corresponds to 5 μm. C, co-transfection of HA-SU1-Bcl11b impairs the transcriptional repression activity of wild-type Bcl11b on the Id2 promoter. The amounts of transfected expression vectors encoding Bcl11b and HA-SU1-Bcl11b (200 or 400 ng) are indicated below. D, SUMO1-Bcl11b interacts with wild-type Bcl11b. Transfections, immunoprecipitations, and immunoblotting were conducted as indicated. The amounts of transfected expression vector encoding Bcl11b and HA-SU1-Bcl11b (200 or 400 ng) are indicated above the immunoblot. E, fusion to SUMO2 abrogates Bcl11b-mediated repression. The amounts of transfected expression vectors encoding Bcl11b and HA-SU2-Bcl11b (200 or 400 ng) are indicated below the figure. Experiments were conducted as described in A. Similar levels of Bcl11b and the HA-SU2-Bcl11b fusion protein were expressed as shown in the blot below the figure. F, SENP1-mediated desumoylation enhances Bcl11b-mediated repression. Expression vectors encoding wild-type Bcl11b or the SUMO-deficient mutant 2R were co-transfected (400 ng of each) with those encoding HA-SUMO1 (800 ng) and the Id2-CAT reporter plasmid (2 μg) with or without a co-transfected expression vector encoding HA-SENP1 (800 ng) as indicated. Note that the expression vector for HA-SUMO1 was co-transfected in all lanes to promote maximum sumoylation of Bcl11b. Bars and error bars in panels A, C, E, and F represent mean relative -fold repression values ±S.E., respectively, (n = 3). Statistical significance throughout the figure was determined using Student's t test (*, p < 0.05; **, p < 0.01).

FIGURE 9.

Prolonged activation of thymocytes results in ubiquitination and degradation of Bcl11b. A, degradation of Bcl11b. Thymocytes were stimulated with P/A as indicated prior to immunoblotting using the anti-Bcl11b and anti-HDAC1 (loading control) antibodies. B, time course of Bcl11b ubiquitination in stimulated thymocytes. Time of P/A treatment, immunoprecipitation, and immunoblotting were as indicated. The immunoprecipitates were probed with anti-ubiquitin (Ub) (upper blot), and the blot was then stripped and probed with an anti-Bcl11b antibody (lower blot). Similar results were obtained in two to four additional independent experiments.

FIGURE 10.

Coordinated regulation of Bcl11b transcriptional activity by reversible phosphorylation and sumoylation in thymocytes. This figure summarizes the relevant findings presented herein. Note that SENPx refers to either SENP1 or SENP2 and perhaps other SENP family members. The term “kinase” refers to Erk1/2 and p38, but other kinases may be implicated in phosphorylation of Bcl11b. Similarly, PPTase refers to the phosphatase PP6, but other phosphatases may be involved in the dephosphorylation of phospho-Bcl11b. Time domains are represented in italicized text, and signaling components between TCR and the MAP kinases Erk1/2 and p38 were omitted for clarity. See discussion for further details of this summary. p-p38, phospho-p38; pErk1/2, phospho-Erk1/2; pPPTase, phosphorylated phosphoprotein phosphatase.