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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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SPAR physically associates with the SCFβ-TRCP complex. A, Plk2-dependent loss of SPAR. HEK293T cells were transfected with 1 μg of pCMV-HA-Plk2 (lane 1), 1 μg of pCMV-myc-SPAR (lane 3), or 1 μg of pCMV-myc-SPAR together with either 1 μg of catalytically inactive pCMV-HA-Plk2D201A (lane 2) or increasing amounts of pCMV-HA-Plk2WT (0.3, 1, or 2 μg) (lanes 4-6). The total amount of transfected DNA was kept constant among all conditions with use of empty vector. Whole cell lysates were immunoblotted with Myc antibody to assess myc-SPAR levels. B, dominant negative versions of Cul1 and β-TRCP stabilize SPAR. pCMV-myc-SPAR (0.5 μg) and pCMV-HA-Plk2D201A/WT (1 μg) (catalytically inactive, lane 1; wild type, lanes 2-9) were co-expressed in HEK293T cells with 2.5 μg of either empty vector, dominant negative Cullins, or dominant negative F-box proteins. Changes in the abundance of myc-SPAR were determined by immunoblotting with anti-Myc antibody. C, F-box protein interaction screen. pCMV-myc-SPAR (0.6 μg), pCMV-HA-Plk2WT/K108M (0.6 μg), and pCMV-DNCul1 (2 μg) were co-expressed as shown with pCMV-GST (lane 2) or the indicated F-box proteins as GST fusions (0.6 μg) (lanes 3-22) in HEK293T cells seeded in 6-well plates. After 24 h, cell extracts were used for GSH-Sepharose pull-down assays, and proteins were immunoblotted with anti-GST and anti-Myc antibodies. Crude lysates were blotted as an input control. D, coimmunoprecipitation of SPAR and Plk2. Extracts of HEK293T cells transfected with pCMV-DNCul1 and pCMV-myc-SPAR (lane 1), pCMV-HA-Plk2 (lane 2), or both (lane 3) were immunoprecipitated using anti-Myc or anti-HA antibodies as indicated. E, formation of a SPAR·Plk2·β-TRCP·Cul1 complex with active Plk2. Lysates from cells transfected with pCMV-myc-SPAR (0.5 μg), pCMV-HA-Plk2WT/D201A (0.5 μg), pCMV-GST-β-TRCP (0.5 μg), and pCMV-FLAG-Cul11-452 (2 μg), as indicated, were incubated with GSH-Sepharose and immunoblotted with anti-Myc, anti-HA, anti-GST, and anti-FLAG antibodies as shown. Lanes 1-4 show lysates (6% of input of the GSH-Sepharose binding reactions).
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fig2: SPAR physically associates with the SCFβ-TRCP complex. A, Plk2-dependent loss of SPAR. HEK293T cells were transfected with 1 μg of pCMV-HA-Plk2 (lane 1), 1 μg of pCMV-myc-SPAR (lane 3), or 1 μg of pCMV-myc-SPAR together with either 1 μg of catalytically inactive pCMV-HA-Plk2D201A (lane 2) or increasing amounts of pCMV-HA-Plk2WT (0.3, 1, or 2 μg) (lanes 4-6). The total amount of transfected DNA was kept constant among all conditions with use of empty vector. Whole cell lysates were immunoblotted with Myc antibody to assess myc-SPAR levels. B, dominant negative versions of Cul1 and β-TRCP stabilize SPAR. pCMV-myc-SPAR (0.5 μg) and pCMV-HA-Plk2D201A/WT (1 μg) (catalytically inactive, lane 1; wild type, lanes 2-9) were co-expressed in HEK293T cells with 2.5 μg of either empty vector, dominant negative Cullins, or dominant negative F-box proteins. Changes in the abundance of myc-SPAR were determined by immunoblotting with anti-Myc antibody. C, F-box protein interaction screen. pCMV-myc-SPAR (0.6 μg), pCMV-HA-Plk2WT/K108M (0.6 μg), and pCMV-DNCul1 (2 μg) were co-expressed as shown with pCMV-GST (lane 2) or the indicated F-box proteins as GST fusions (0.6 μg) (lanes 3-22) in HEK293T cells seeded in 6-well plates. After 24 h, cell extracts were used for GSH-Sepharose pull-down assays, and proteins were immunoblotted with anti-GST and anti-Myc antibodies. Crude lysates were blotted as an input control. D, coimmunoprecipitation of SPAR and Plk2. Extracts of HEK293T cells transfected with pCMV-DNCul1 and pCMV-myc-SPAR (lane 1), pCMV-HA-Plk2 (lane 2), or both (lane 3) were immunoprecipitated using anti-Myc or anti-HA antibodies as indicated. E, formation of a SPAR·Plk2·β-TRCP·Cul1 complex with active Plk2. Lysates from cells transfected with pCMV-myc-SPAR (0.5 μg), pCMV-HA-Plk2WT/D201A (0.5 μg), pCMV-GST-β-TRCP (0.5 μg), and pCMV-FLAG-Cul11-452 (2 μg), as indicated, were incubated with GSH-Sepharose and immunoblotted with anti-Myc, anti-HA, anti-GST, and anti-FLAG antibodies as shown. Lanes 1-4 show lysates (6% of input of the GSH-Sepharose binding reactions).

Mentions: SPAR Physically Associates with the SCFβ-TRCP Complex—To further explore the idea that SPAR turnover is regulated by an SCF E3 Ub-ligase, we established a system in cultured HEK293T cells that recapitulates Plk2-dependent SPAR degradation. This system facilitated biochemical studies that were otherwise limited by the physical properties of dendritic spines and allowed the use of molecular reagents previously developed for study of the human SCF pathway (24). We found that expression of SPAR alone (as an Myc-tagged fusion protein) led to its accumulation in HEK293T cells (Fig. 2A, lane 3). In contrast, co-expression with Plk2 (but not a catalytically inactive mutant Plk2D201A) promoted the degradation of myc-SPAR (Fig. 2, A, lane 2 compared with lanes 4-6, and B, lanes 1 and 2). Co-expression of SPAR with wild type Plk2 correlated with the appearance of a slower mobility form of SPAR (presumably phosphorylated) that is sensitive to Plk2-induced degradation (Fig. 2A, myc-SPAR-P; see also Fig. 2, B and C). Our recapitulation of Plk2-dependent SPAR turnover in HEK293T cells provides a convenient system in which to search for components of the Plk2-dependent degradation pathway.


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)

SPAR physically associates with the SCFβ-TRCP complex. A, Plk2-dependent loss of SPAR. HEK293T cells were transfected with 1 μg of pCMV-HA-Plk2 (lane 1), 1 μg of pCMV-myc-SPAR (lane 3), or 1 μg of pCMV-myc-SPAR together with either 1 μg of catalytically inactive pCMV-HA-Plk2D201A (lane 2) or increasing amounts of pCMV-HA-Plk2WT (0.3, 1, or 2 μg) (lanes 4-6). The total amount of transfected DNA was kept constant among all conditions with use of empty vector. Whole cell lysates were immunoblotted with Myc antibody to assess myc-SPAR levels. B, dominant negative versions of Cul1 and β-TRCP stabilize SPAR. pCMV-myc-SPAR (0.5 μg) and pCMV-HA-Plk2D201A/WT (1 μg) (catalytically inactive, lane 1; wild type, lanes 2-9) were co-expressed in HEK293T cells with 2.5 μg of either empty vector, dominant negative Cullins, or dominant negative F-box proteins. Changes in the abundance of myc-SPAR were determined by immunoblotting with anti-Myc antibody. C, F-box protein interaction screen. pCMV-myc-SPAR (0.6 μg), pCMV-HA-Plk2WT/K108M (0.6 μg), and pCMV-DNCul1 (2 μg) were co-expressed as shown with pCMV-GST (lane 2) or the indicated F-box proteins as GST fusions (0.6 μg) (lanes 3-22) in HEK293T cells seeded in 6-well plates. After 24 h, cell extracts were used for GSH-Sepharose pull-down assays, and proteins were immunoblotted with anti-GST and anti-Myc antibodies. Crude lysates were blotted as an input control. D, coimmunoprecipitation of SPAR and Plk2. Extracts of HEK293T cells transfected with pCMV-DNCul1 and pCMV-myc-SPAR (lane 1), pCMV-HA-Plk2 (lane 2), or both (lane 3) were immunoprecipitated using anti-Myc or anti-HA antibodies as indicated. E, formation of a SPAR·Plk2·β-TRCP·Cul1 complex with active Plk2. Lysates from cells transfected with pCMV-myc-SPAR (0.5 μg), pCMV-HA-Plk2WT/D201A (0.5 μg), pCMV-GST-β-TRCP (0.5 μg), and pCMV-FLAG-Cul11-452 (2 μg), as indicated, were incubated with GSH-Sepharose and immunoblotted with anti-Myc, anti-HA, anti-GST, and anti-FLAG antibodies as shown. Lanes 1-4 show lysates (6% of input of the GSH-Sepharose binding reactions).
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Related In: Results  -  Collection

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fig2: SPAR physically associates with the SCFβ-TRCP complex. A, Plk2-dependent loss of SPAR. HEK293T cells were transfected with 1 μg of pCMV-HA-Plk2 (lane 1), 1 μg of pCMV-myc-SPAR (lane 3), or 1 μg of pCMV-myc-SPAR together with either 1 μg of catalytically inactive pCMV-HA-Plk2D201A (lane 2) or increasing amounts of pCMV-HA-Plk2WT (0.3, 1, or 2 μg) (lanes 4-6). The total amount of transfected DNA was kept constant among all conditions with use of empty vector. Whole cell lysates were immunoblotted with Myc antibody to assess myc-SPAR levels. B, dominant negative versions of Cul1 and β-TRCP stabilize SPAR. pCMV-myc-SPAR (0.5 μg) and pCMV-HA-Plk2D201A/WT (1 μg) (catalytically inactive, lane 1; wild type, lanes 2-9) were co-expressed in HEK293T cells with 2.5 μg of either empty vector, dominant negative Cullins, or dominant negative F-box proteins. Changes in the abundance of myc-SPAR were determined by immunoblotting with anti-Myc antibody. C, F-box protein interaction screen. pCMV-myc-SPAR (0.6 μg), pCMV-HA-Plk2WT/K108M (0.6 μg), and pCMV-DNCul1 (2 μg) were co-expressed as shown with pCMV-GST (lane 2) or the indicated F-box proteins as GST fusions (0.6 μg) (lanes 3-22) in HEK293T cells seeded in 6-well plates. After 24 h, cell extracts were used for GSH-Sepharose pull-down assays, and proteins were immunoblotted with anti-GST and anti-Myc antibodies. Crude lysates were blotted as an input control. D, coimmunoprecipitation of SPAR and Plk2. Extracts of HEK293T cells transfected with pCMV-DNCul1 and pCMV-myc-SPAR (lane 1), pCMV-HA-Plk2 (lane 2), or both (lane 3) were immunoprecipitated using anti-Myc or anti-HA antibodies as indicated. E, formation of a SPAR·Plk2·β-TRCP·Cul1 complex with active Plk2. Lysates from cells transfected with pCMV-myc-SPAR (0.5 μg), pCMV-HA-Plk2WT/D201A (0.5 μg), pCMV-GST-β-TRCP (0.5 μg), and pCMV-FLAG-Cul11-452 (2 μg), as indicated, were incubated with GSH-Sepharose and immunoblotted with anti-Myc, anti-HA, anti-GST, and anti-FLAG antibodies as shown. Lanes 1-4 show lysates (6% of input of the GSH-Sepharose binding reactions).
Mentions: SPAR Physically Associates with the SCFβ-TRCP Complex—To further explore the idea that SPAR turnover is regulated by an SCF E3 Ub-ligase, we established a system in cultured HEK293T cells that recapitulates Plk2-dependent SPAR degradation. This system facilitated biochemical studies that were otherwise limited by the physical properties of dendritic spines and allowed the use of molecular reagents previously developed for study of the human SCF pathway (24). We found that expression of SPAR alone (as an Myc-tagged fusion protein) led to its accumulation in HEK293T cells (Fig. 2A, lane 3). In contrast, co-expression with Plk2 (but not a catalytically inactive mutant Plk2D201A) promoted the degradation of myc-SPAR (Fig. 2, A, lane 2 compared with lanes 4-6, and B, lanes 1 and 2). Co-expression of SPAR with wild type Plk2 correlated with the appearance of a slower mobility form of SPAR (presumably phosphorylated) that is sensitive to Plk2-induced degradation (Fig. 2A, myc-SPAR-P; see also Fig. 2, B and C). Our recapitulation of Plk2-dependent SPAR turnover in HEK293T cells provides a convenient system in which to search for components of the Plk2-dependent degradation pathway.

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