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. 2014 Jul 16;34(29):9574-89.
doi: 10.1523/JNEUROSCI.4302-13.2014.

CREB SUMOylation by the E3 ligase PIAS1 enhances spatial memory

Yan-Chu Chen  1 , Wei-Lun Hsu  2 , Yun-Li Ma  2 , Derek J C Tai  2 , Eminy H Y Lee  3
Affiliations

CREB SUMOylation by the E3 ligase PIAS1 enhances spatial memory

Yan-Chu Chen et al. J Neurosci. .

Abstract

cAMP-responsive element binding protein (CREB) phosphorylation and signaling plays an important role in long-term memory formation, but other posttranslational modifications of CREB are less known. Here, we found that CREB1Δ, the short isoform of CREB, could be sumoylated by the small ubiquitin-like modifier (SUMO) E3 ligase protein inhibitor of activated STAT1 (PIAS1) at Lys271 and Lys290 and PIAS1 SUMOylation of CREB1Δ increased the expression level of CREB1Δ. CREB1Δ could also be sumoylated by other PIAS family proteins, but not by the E3 ligases RanBP2 and Pc2 or by the E2 ligase Ubc9. Furthermore, water maze training increased the level of endogenous CREB SUMOylation in rat CA1 neurons determined by in vitro SUMOylation assay, but this effect was not observed in other brain areas. Moreover, transduction of Lenti-CREBWT to rat CA1 area facilitated, whereas transduction of Lenti-CREB double sumo-mutant (CREBK271RK290R) impaired, spatial learning and memory performance. Transduction of Lenti-CREBWT-SUMO1 fusion vector to rat CA1 area showed a more significant effect in enhancing spatial learning and memory and CREB SUMOylation. Lenti-CREBWT transduction increased, whereas Lenti-CREBK271RK290R transduction decreased, CREB DNA binding to the brain-derived neurotrophic factor (bdnf) promoter and decreased bdnf mRNA expression. Knock-down of PIAS1 expression in CA1 area by PIAS1 siRNA transfection impaired spatial learning and memory and decreased endogenous CREB SUMOylation. In addition, CREB SUMOylation was CREB phosphorylation dependent and lasted longer. Therefore, CREB phosphorylation may be responsible for signal transduction during the early phase of long-term memory formation, whereas CREB SUMOylation sustains long-term memory.

Keywords: CREB; PIAS1; SUMOylation; phosphorylation; spatial learning and memory.

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Figures

Figure 1.
Figure 1.
CREB is sumoylated by PIAS1 in vitro and in HEK293T cells. A, Alignment and comparison of CREB1α and CREB1Δ isoforms showing that CREB1Δ is 14 aa shorter than that of CREB1α (aa 88–101), and that Ser119 of CREB1Δ corresponds to Ser133 of CREB1α. B, In vitro SUMOylation assay and Western blot showing PIAS1 SUMOylation of CREB. “a” and “b” indicate the sumoylated form of the CREB bands. C, Co-IP experiment showing the association between PIAS1 and CREB in HEK293T cells with or without the cotransfection of Myc-SUMO1 or Myc-SUMO1 (ΔGG). The quantified results are shown on the bottom. D, Transfection of different plasmids to HEK293T cells and Western blot showing PIAS1 SUMOylation of CREB in HEK293T cells. E, Confirmation of PIAS1 SUMOylation of CREB in HEK293T cells by IP and Western blot against CREB and SUMO1. endo-CREB, Endogenous CREB; ΔGG, C-terminal diglycine motif truncated SUMO1. Data are expressed as mean ± SEM. **p < 0.05; **p < 0.01; #p < 0.001. Experiments were performed in triplicate.
Figure 2.
Figure 2.
Identification of the major SUMO acceptors on CREB. A, Examination of PIAS1 SUMOylation of CREB at Lys109, Lys122, Lys141, Lys271, and Lys278 in HEK293T cells. B, Examination of PIAS1 SUMOylation of CREB at Lys289, Lys290, Lys291, and Lys295 in HEK293T cells. C, Examination of PIAS1 SUMOylation of CREB at Lys309, Lys316, Lys319, and Lys325 in HEK293T cells. D, Confirmation of PIAS1 SUMOylation of CREB at Lys271 and Lys290 in HEK293T cells. The quantified result is shown on the right. E, PIAS1 overexpression increased the expression level of CREB in HEK293T cells. The quantified result over time is shown below. CHX, Cycloheximide. Data are shown as mean ± SEM. F, CREB SUMOylation by PIAS family proteins, other E3 ligases, and E2 ligase in HEK293T cells. The quantified result is shown on the right. ΔFG, Partial sequence of full-length RanBP2. Data are expressed as mean ± SEM. *p < 0.05; #p < 0.001. Experiments were performed in duplicate or triplicate.
Figure 3.
Figure 3.
CREB is sumoylated by PIAS1 in the hippocampus. A, Co-IP showing that PIAS1 is associated with CREB and vice versa in rat hippocampus. Identification of CREB sumo acceptors in rat hippocampus by Western blot against CREB (B) and SUMO1 (C). The quantified results are shown on the right. D, Confocal images showing EGFP-CREB transfection and expression in the CA1 area at different magnifications. Cells that show both green fluorescence (EGFP) and blue fluorescence (DAPI) are cells successfully transfected with the plasmid. Red arrows indicate the area of transfection. Scale bars: top, 500 μm; bottom, 100 μm. E, NMDA was injected into rat CA1 area bilaterally (1.2 μg/μl, 0.7 μl each side) and in vitro SUMOylation for CREB was performed 30 min later. In one group, the SUMO1 mutant protein was added to the reaction and, in another group, the ligase-deficient mutant Flag-PIAS1W372A plasmid was transfected. Effect of NMDA injection was confirmed by elevated level of pCREB from Western blot in a separate analysis. Flag-PIAS1W372A transfection was confirmed by Western blot against the Flag tag. n = 5 each group. The quantified result is shown on the right. F, Subcellular distribution of PIAS1, CREB, and the sumoylated form of CREB in the cytosol and nucleus fractions from the rat CA1 tissue. Data are expressed as mean ± SEM. **p < 0.01; #p < 0.001. Experiments were performed in duplicate or triplicate.
Figure 4.
Figure 4.
Time course change of CREB phosphorylation and CREB SUMOylation in rat hippocampus. Naive rats were randomly assigned to the swim control group or subjected to spatial training for 1 d and their CA1 tissue was dissected out for determination of CREB phosphorylation (A) and endogenous CREB SUMOylation (B). The quantified results are shown in C. Separate groups of animals were divided into the swim control group or subjected to spatial training for 2 d and their CA1 tissue was dissected out for determination of CREB phosphorylation (D) and endogenous CREB SUMOylation (E). The quantified results are shown in F. Two other groups of animals were divided into the swim control group or subjected to spatial training for 5 d and their CA1 tissue was dissected out for determination of CREB phosphorylation (G) and endogenous CREB SUMOylation (H). The quantified results are shown in I. Endogenous CREB SUMOylation in the striatum (J) and amygdala (K) from the same animals (trained for 5 d) and the quantified results are shown. n = 5 each group. Data are expressed as mean ± SEM. #p < 0.001.
Figure 5.
Figure 5.
CREB SUMOylation enhances spatial learning and memory and increases CREB DNA binding and bdnf mRNA expression. Effects of Lenti-CREBWT and Lenti-CREB sumo-mutant transduction to rat CA1 area on spatial acquisition (A), probe trial performance (B), CREB SUMOylation and CREB DNA binding (C). n = 7 each group. D, ChIP PCR (top) and ChIP qPCR (bottom) showing the effects of Flag-CREBWT and Flag-CREB sumo-mutant transfection on CREB DNA binding to the bdnf promoter. E, Quantitative PCR showing the effects of Flag-CREBWT and Flag-CREB sumo-mutant transfection on bndf mRNA level. F, Effect of Lenti-CREBWT and Lenti-CREB sumo-mutant transduction to rat CA1 area on visible platform learning (n = 4 each group). Data are expressed as mean ± SEM. Biochemical experiments were performed in duplicate. *p < 0.05; **p < 0.01; #p < 0.001.
Figure 6.
Figure 6.
Lenti-CREB-SUMO1 fusion vector transduction enhances spatial learning and memory and increases CREB SUMOylation. Effects of Lenti-CREBWT and Lenti-CREB-SUMO1 fusion vector transduction to the rat CA1 area on spatial acquisition (A), probe trial performance (B), and CREB SUMOylation (C) are shown. The quantified result of CREB SUMOylation is shown in D. n = 8 each group. Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; #p < 0.001.
Figure 7.
Figure 7.
PIAS1 siRNA transfection impairs water maze performance and decreases CREB SUMOylation. Effects of PIAS1 siRNA transfection to the rat CA1 area on spatial acquisition (A), probe trial performance (B), endogenous CREB SUMOylation and the expression of different PIAS family proteins (C) are shown. The quantified result of CREB SUMOylation is shown in D. n = 7 each group. Data are expressed as mean ± SEM. *p < 0.05; #p < 0.001.
Figure 8.
Figure 8.
CREB SUMOylation is CREB phosphorylation dependent. A, Flag-CREBWT or Flag-CREBS119A plasmid was transfected to rat CA1 area, with or without NMDA coinjection, and CREB SUMOylation and CREB phosphorylation were determined by Western blot. The quantified results are shown in B. C, Flag-CREBWT or Flag-CREBK271RK290R was transfected to rat CA1 area, with or without NMDA coinjection, and CREB SUMOylation and CREB phosphorylation were determined by Western blot. The quantified results are shown in D. n = 3 each group. Data are shown as mean ± SEM. *p < 0.05; **p < 0.01; #p < 0.001.
Figure 9.
Figure 9.
Schematic diagram showing the relationships among spatial training, PIAS1 expression, CREB SUMOylation, BDNF expression, and spatial memory formation. Spatial training increases PIAS1 expression through activation of the MAPK/ERK signaling cascade (Liu et al., 2013). Increased PIAS1 expression enhances CREB SUMOylation and CREB DNA binding to the gene promoter that upregulates BDNF expression. Increased BDNF expression facilitates spatial memory formation.
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