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Regulation of postsynaptic RapGAP SPAR by Polo-like kinase 2 and the SCFbeta-TRCP ubiquitin ligase in hippocampal neurons.

Ang XL, Seeburg DP, Sheng M, Harper JW - J. Biol. Chem. (2008)

Bottom Line: In the presence of Plk2, SPAR physically associated with the SCF(beta-TRCP) complex through a canonical phosphodegron.In hippocampal neurons, disruption of the SCF(beta-TRCP) complex by overexpression of dominant interfering beta-TRCP or Cul1 constructs prevented Plk2-dependent degradation of SPAR.Our results identify a specific E3 ubiquitin ligase that mediates degradation of a key postsynaptic regulator of synaptic morphology and function.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
The ubiquitin-proteasome pathway (UPP) regulates synaptic function, but little is known about specific UPP targets and mechanisms in mammalian synapses. We report here that the SCF(beta-TRCP) complex, a multisubunit E3 ubiquitin ligase, targets the postsynaptic spine-associated Rap GTPase activating protein (SPAR) for degradation in neurons. SPAR degradation by SCF(beta-TRCP) depended on the activity-inducible protein kinase Polo-like kinase 2 (Plk2). In the presence of Plk2, SPAR physically associated with the SCF(beta-TRCP) complex through a canonical phosphodegron. In hippocampal neurons, disruption of the SCF(beta-TRCP) complex by overexpression of dominant interfering beta-TRCP or Cul1 constructs prevented Plk2-dependent degradation of SPAR. Our results identify a specific E3 ubiquitin ligase that mediates degradation of a key postsynaptic regulator of synaptic morphology and function.

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SCFβ-TRCP regulates Plk2-dependent SPAR abundance, turnover, and promotes its ubiquitination. A and B, depletion of β-TRCP by RNAi protects GFP-SPAR from degradation in individual HEK293T cells. The indicated plasmids were transfected with pCMV-RFP as a co-transfection marker into HEK293T cells. After 96 h, cells were fixed and imaged for GFP-SPAR and RFP expression (A) and quantified as the percentage of RFP expressing cells that also expressed GFP-SPAR (B). Values represent the mean ± S.E. from three independent experiments, derived from analysis of 400 cells per experiment per condition (n = 1200 cells per condition). **, p < 0.01; NS, not significant; one-way analysis of variance, compared with control condition (pRSP+Plk2D/A). C and D, depletion of β-TRCP by RNAi stabilizes SPAR abundance and turnover in the presence of Plk2 activity. HEK293T cells were transfected with vectors expressing myc-SPAR (0.5 μg), WT, or catalytically inactive HA-Plk2 (D/A) (1 μg), and the indicated shRNA vector carrying a puromycin resistance selection marker (2.5 μg). 36 h post-transfection, cells were incubated with media containing 1 μg/ml puromycin to enrich for shRNA expression. Extracts were subsequently examined by immunoblotting 96 h post-transfection as indicated. Endogenous Cdc25A was probed to control for successful knockdown of β-TRCP. In panel D, HEK293T cells were transfected in an identical fashion to panel C, except that 24 h post-transfection the transfected cells were split among 5 wells in puromycin-containing media. After 48 h of puromycin selection, cells were treated with 25 μg/ml cycloheximide (CHX) and harvested at the indicated times before immunoblotting. E, expression of β-TRCP promotes Plk2-mediated SPAR ubiquitination. HEK293T cells were transfected with myc-SPAR (1 μg), His-Ub (1 μg), HA-Plk2 (1.5 μg, wild type or D201A), and GST or GST-β-TRCP (4.5 μg). Twenty hours post-transfection cells were treated with 25 μm MG-132 for 5 h and lysed in buffer containing 10 mm N-ethylmaleimide. Myc-SPAR was purified with c-Myc 9E10-agarose, resolved on 6% Tris-glycine SDS-PAGE gel, and immunoblotted (IB) with anti-Myc antibodies. IP, immunoprecipitates.
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fig3: SCFβ-TRCP regulates Plk2-dependent SPAR abundance, turnover, and promotes its ubiquitination. A and B, depletion of β-TRCP by RNAi protects GFP-SPAR from degradation in individual HEK293T cells. The indicated plasmids were transfected with pCMV-RFP as a co-transfection marker into HEK293T cells. After 96 h, cells were fixed and imaged for GFP-SPAR and RFP expression (A) and quantified as the percentage of RFP expressing cells that also expressed GFP-SPAR (B). Values represent the mean ± S.E. from three independent experiments, derived from analysis of 400 cells per experiment per condition (n = 1200 cells per condition). **, p < 0.01; NS, not significant; one-way analysis of variance, compared with control condition (pRSP+Plk2D/A). C and D, depletion of β-TRCP by RNAi stabilizes SPAR abundance and turnover in the presence of Plk2 activity. HEK293T cells were transfected with vectors expressing myc-SPAR (0.5 μg), WT, or catalytically inactive HA-Plk2 (D/A) (1 μg), and the indicated shRNA vector carrying a puromycin resistance selection marker (2.5 μg). 36 h post-transfection, cells were incubated with media containing 1 μg/ml puromycin to enrich for shRNA expression. Extracts were subsequently examined by immunoblotting 96 h post-transfection as indicated. Endogenous Cdc25A was probed to control for successful knockdown of β-TRCP. In panel D, HEK293T cells were transfected in an identical fashion to panel C, except that 24 h post-transfection the transfected cells were split among 5 wells in puromycin-containing media. After 48 h of puromycin selection, cells were treated with 25 μg/ml cycloheximide (CHX) and harvested at the indicated times before immunoblotting. E, expression of β-TRCP promotes Plk2-mediated SPAR ubiquitination. HEK293T cells were transfected with myc-SPAR (1 μg), His-Ub (1 μg), HA-Plk2 (1.5 μg, wild type or D201A), and GST or GST-β-TRCP (4.5 μg). Twenty hours post-transfection cells were treated with 25 μm MG-132 for 5 h and lysed in buffer containing 10 mm N-ethylmaleimide. Myc-SPAR was purified with c-Myc 9E10-agarose, resolved on 6% Tris-glycine SDS-PAGE gel, and immunoblotted (IB) with anti-Myc antibodies. IP, immunoprecipitates.

Mentions: To directly examine whether β-TRCP proteins are required for Plk2-dependent SPAR turnover, we took advantage of a shRNA vector (shβ-TRCP) that is capable of suppressing protein expression of both human β-TRCP1 and β-TRCP2. This hairpin sequence and this particular shRNA vector have been validated for numerous β-TRCP substrates (14, 19, 26). Cells were transfected with expression constructs for GFP-SPAR and red fluorescent protein (RFP) to mark transfected cells and simultaneously transfected with shβ-TRCP or control vector (pRSP) in the presence of active Plk2 or a catalytically inactive version, Plk2D201A (Fig. 3A). We subsequently visualized cells for the presence of GFP-SPAR in RFP-positive cells (Fig. 3A). RFP-positive cells expressing Plk2, but not those expressing catalytically inactive Plk2D201A, displayed very low levels of GFP-SPAR in the presence of the control shRNA vector pRSP (Fig. 3A, quantified in B). In contrast, RFP-positive cells transfected with shβ-TRCP contained high levels of GFP-SPAR despite the co-transfection of active Plk2 (Fig. 3A, quantified in B). Thus, depletion of β-TRCP by RNAi substantially protected GFP-SPAR from Plk2-dependent degradation. Immunoblotting of transfected cell lysates confirmed that Plk2-induced degradation of myc-SPAR requires β-TRCP (Fig. 3C). As expected, Plk2 promoted loss of myc-SPAR in cells expressing a control shRNA that targets GFP (shGFP) (lanes 1 and 2). In contrast, myc-SPAR was not efficiently degraded in cells depleted of β-TRCP despite the presence of active Plk2 (lane 4). Furthermore, depletion of β-TRCP led to accumulation of Cdc25A, a known target of SCFβ-TRCP used here as a positive control (14, 18).


Regulation of postsynaptic RapGAP SPAR by Polo-like kinase 2 and the SCFbeta-TRCP ubiquitin ligase in hippocampal neurons.

Ang XL, Seeburg DP, Sheng M, Harper JW - J. Biol. Chem. (2008)

SCFβ-TRCP regulates Plk2-dependent SPAR abundance, turnover, and promotes its ubiquitination. A and B, depletion of β-TRCP by RNAi protects GFP-SPAR from degradation in individual HEK293T cells. The indicated plasmids were transfected with pCMV-RFP as a co-transfection marker into HEK293T cells. After 96 h, cells were fixed and imaged for GFP-SPAR and RFP expression (A) and quantified as the percentage of RFP expressing cells that also expressed GFP-SPAR (B). Values represent the mean ± S.E. from three independent experiments, derived from analysis of 400 cells per experiment per condition (n = 1200 cells per condition). **, p < 0.01; NS, not significant; one-way analysis of variance, compared with control condition (pRSP+Plk2D/A). C and D, depletion of β-TRCP by RNAi stabilizes SPAR abundance and turnover in the presence of Plk2 activity. HEK293T cells were transfected with vectors expressing myc-SPAR (0.5 μg), WT, or catalytically inactive HA-Plk2 (D/A) (1 μg), and the indicated shRNA vector carrying a puromycin resistance selection marker (2.5 μg). 36 h post-transfection, cells were incubated with media containing 1 μg/ml puromycin to enrich for shRNA expression. Extracts were subsequently examined by immunoblotting 96 h post-transfection as indicated. Endogenous Cdc25A was probed to control for successful knockdown of β-TRCP. In panel D, HEK293T cells were transfected in an identical fashion to panel C, except that 24 h post-transfection the transfected cells were split among 5 wells in puromycin-containing media. After 48 h of puromycin selection, cells were treated with 25 μg/ml cycloheximide (CHX) and harvested at the indicated times before immunoblotting. E, expression of β-TRCP promotes Plk2-mediated SPAR ubiquitination. HEK293T cells were transfected with myc-SPAR (1 μg), His-Ub (1 μg), HA-Plk2 (1.5 μg, wild type or D201A), and GST or GST-β-TRCP (4.5 μg). Twenty hours post-transfection cells were treated with 25 μm MG-132 for 5 h and lysed in buffer containing 10 mm N-ethylmaleimide. Myc-SPAR was purified with c-Myc 9E10-agarose, resolved on 6% Tris-glycine SDS-PAGE gel, and immunoblotted (IB) with anti-Myc antibodies. IP, immunoprecipitates.
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fig3: SCFβ-TRCP regulates Plk2-dependent SPAR abundance, turnover, and promotes its ubiquitination. A and B, depletion of β-TRCP by RNAi protects GFP-SPAR from degradation in individual HEK293T cells. The indicated plasmids were transfected with pCMV-RFP as a co-transfection marker into HEK293T cells. After 96 h, cells were fixed and imaged for GFP-SPAR and RFP expression (A) and quantified as the percentage of RFP expressing cells that also expressed GFP-SPAR (B). Values represent the mean ± S.E. from three independent experiments, derived from analysis of 400 cells per experiment per condition (n = 1200 cells per condition). **, p < 0.01; NS, not significant; one-way analysis of variance, compared with control condition (pRSP+Plk2D/A). C and D, depletion of β-TRCP by RNAi stabilizes SPAR abundance and turnover in the presence of Plk2 activity. HEK293T cells were transfected with vectors expressing myc-SPAR (0.5 μg), WT, or catalytically inactive HA-Plk2 (D/A) (1 μg), and the indicated shRNA vector carrying a puromycin resistance selection marker (2.5 μg). 36 h post-transfection, cells were incubated with media containing 1 μg/ml puromycin to enrich for shRNA expression. Extracts were subsequently examined by immunoblotting 96 h post-transfection as indicated. Endogenous Cdc25A was probed to control for successful knockdown of β-TRCP. In panel D, HEK293T cells were transfected in an identical fashion to panel C, except that 24 h post-transfection the transfected cells were split among 5 wells in puromycin-containing media. After 48 h of puromycin selection, cells were treated with 25 μg/ml cycloheximide (CHX) and harvested at the indicated times before immunoblotting. E, expression of β-TRCP promotes Plk2-mediated SPAR ubiquitination. HEK293T cells were transfected with myc-SPAR (1 μg), His-Ub (1 μg), HA-Plk2 (1.5 μg, wild type or D201A), and GST or GST-β-TRCP (4.5 μg). Twenty hours post-transfection cells were treated with 25 μm MG-132 for 5 h and lysed in buffer containing 10 mm N-ethylmaleimide. Myc-SPAR was purified with c-Myc 9E10-agarose, resolved on 6% Tris-glycine SDS-PAGE gel, and immunoblotted (IB) with anti-Myc antibodies. IP, immunoprecipitates.
Mentions: To directly examine whether β-TRCP proteins are required for Plk2-dependent SPAR turnover, we took advantage of a shRNA vector (shβ-TRCP) that is capable of suppressing protein expression of both human β-TRCP1 and β-TRCP2. This hairpin sequence and this particular shRNA vector have been validated for numerous β-TRCP substrates (14, 19, 26). Cells were transfected with expression constructs for GFP-SPAR and red fluorescent protein (RFP) to mark transfected cells and simultaneously transfected with shβ-TRCP or control vector (pRSP) in the presence of active Plk2 or a catalytically inactive version, Plk2D201A (Fig. 3A). We subsequently visualized cells for the presence of GFP-SPAR in RFP-positive cells (Fig. 3A). RFP-positive cells expressing Plk2, but not those expressing catalytically inactive Plk2D201A, displayed very low levels of GFP-SPAR in the presence of the control shRNA vector pRSP (Fig. 3A, quantified in B). In contrast, RFP-positive cells transfected with shβ-TRCP contained high levels of GFP-SPAR despite the co-transfection of active Plk2 (Fig. 3A, quantified in B). Thus, depletion of β-TRCP by RNAi substantially protected GFP-SPAR from Plk2-dependent degradation. Immunoblotting of transfected cell lysates confirmed that Plk2-induced degradation of myc-SPAR requires β-TRCP (Fig. 3C). As expected, Plk2 promoted loss of myc-SPAR in cells expressing a control shRNA that targets GFP (shGFP) (lanes 1 and 2). In contrast, myc-SPAR was not efficiently degraded in cells depleted of β-TRCP despite the presence of active Plk2 (lane 4). Furthermore, depletion of β-TRCP led to accumulation of Cdc25A, a known target of SCFβ-TRCP used here as a positive control (14, 18).

Bottom Line: In the presence of Plk2, SPAR physically associated with the SCF(beta-TRCP) complex through a canonical phosphodegron.In hippocampal neurons, disruption of the SCF(beta-TRCP) complex by overexpression of dominant interfering beta-TRCP or Cul1 constructs prevented Plk2-dependent degradation of SPAR.Our results identify a specific E3 ubiquitin ligase that mediates degradation of a key postsynaptic regulator of synaptic morphology and function.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
The ubiquitin-proteasome pathway (UPP) regulates synaptic function, but little is known about specific UPP targets and mechanisms in mammalian synapses. We report here that the SCF(beta-TRCP) complex, a multisubunit E3 ubiquitin ligase, targets the postsynaptic spine-associated Rap GTPase activating protein (SPAR) for degradation in neurons. SPAR degradation by SCF(beta-TRCP) depended on the activity-inducible protein kinase Polo-like kinase 2 (Plk2). In the presence of Plk2, SPAR physically associated with the SCF(beta-TRCP) complex through a canonical phosphodegron. In hippocampal neurons, disruption of the SCF(beta-TRCP) complex by overexpression of dominant interfering beta-TRCP or Cul1 constructs prevented Plk2-dependent degradation of SPAR. Our results identify a specific E3 ubiquitin ligase that mediates degradation of a key postsynaptic regulator of synaptic morphology and function.

Show MeSH
Related in: MedlinePlus