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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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Plk2-induced SPAR degradation requires a Cul1-based E3 Ubligase. A-C, dominant negative Cul1 constructs block Plk2-dependent loss of SPAR in hippocampal neurons. Dissociated rat hippocampal neurons (DIV16) were transfected with dominant negative Cullin plasmids (B and C) or a control plasmid (A) and super-infected 2 days later with FLAG-tagged Plk2 driven by Sindbis virus (Sin-Plk2). Neurons were fixed ∼18 h post-infection and immunostained for endogenous SPAR and infected Plk2. Transfected cells were identified during image acquisition by the presence of a co-transfected “fill” protein (GFP, seen in the first column of images). SPAR (green) and Plk2 (red) were pseudo-colored for illustrative purposes after image analysis. Arrows point to cells that are both transfected and infected; arrowheads point to cells that are infected only. Yellow indicates the presence of both SPAR and Plk2 staining. D and E, quantification of SPAR immunostaining in somatic and proximal dendritic regions as integrated immunofluorescence intensity per area in cells transfected with indicated plasmids and/or infected with Plk2 Sindbis virus (Sin-Plk2), normalized to nearby untransfected (untr) cells. Values represent the mean ± S.E., n > 17 cells for all conditions, ***, p < 0.001, Mann-Whitney test (E).
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fig1: Plk2-induced SPAR degradation requires a Cul1-based E3 Ubligase. A-C, dominant negative Cul1 constructs block Plk2-dependent loss of SPAR in hippocampal neurons. Dissociated rat hippocampal neurons (DIV16) were transfected with dominant negative Cullin plasmids (B and C) or a control plasmid (A) and super-infected 2 days later with FLAG-tagged Plk2 driven by Sindbis virus (Sin-Plk2). Neurons were fixed ∼18 h post-infection and immunostained for endogenous SPAR and infected Plk2. Transfected cells were identified during image acquisition by the presence of a co-transfected “fill” protein (GFP, seen in the first column of images). SPAR (green) and Plk2 (red) were pseudo-colored for illustrative purposes after image analysis. Arrows point to cells that are both transfected and infected; arrowheads point to cells that are infected only. Yellow indicates the presence of both SPAR and Plk2 staining. D and E, quantification of SPAR immunostaining in somatic and proximal dendritic regions as integrated immunofluorescence intensity per area in cells transfected with indicated plasmids and/or infected with Plk2 Sindbis virus (Sin-Plk2), normalized to nearby untransfected (untr) cells. Values represent the mean ± S.E., n > 17 cells for all conditions, ***, p < 0.001, Mann-Whitney test (E).

Mentions: Cultured hippocampal neurons (16 days in vitro (DIV16)) were transfected with DNCul1 and then super-infected 2 days later with Sindbis virus driving expression of FLAG-tagged Plk2 for ∼18 h to promote degradation of endogenous SPAR (6). We infected at a titer that resulted in ∼10% infection rate of cells already transfected with DNCul1 (Fig. 1, A-C). The option of co-transfecting plasmids driving Plk2 expression was precluded by the low Plk2 expression achievable using this method.6


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)

Plk2-induced SPAR degradation requires a Cul1-based E3 Ubligase. A-C, dominant negative Cul1 constructs block Plk2-dependent loss of SPAR in hippocampal neurons. Dissociated rat hippocampal neurons (DIV16) were transfected with dominant negative Cullin plasmids (B and C) or a control plasmid (A) and super-infected 2 days later with FLAG-tagged Plk2 driven by Sindbis virus (Sin-Plk2). Neurons were fixed ∼18 h post-infection and immunostained for endogenous SPAR and infected Plk2. Transfected cells were identified during image acquisition by the presence of a co-transfected “fill” protein (GFP, seen in the first column of images). SPAR (green) and Plk2 (red) were pseudo-colored for illustrative purposes after image analysis. Arrows point to cells that are both transfected and infected; arrowheads point to cells that are infected only. Yellow indicates the presence of both SPAR and Plk2 staining. D and E, quantification of SPAR immunostaining in somatic and proximal dendritic regions as integrated immunofluorescence intensity per area in cells transfected with indicated plasmids and/or infected with Plk2 Sindbis virus (Sin-Plk2), normalized to nearby untransfected (untr) cells. Values represent the mean ± S.E., n > 17 cells for all conditions, ***, p < 0.001, Mann-Whitney test (E).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2570879&req=5

fig1: Plk2-induced SPAR degradation requires a Cul1-based E3 Ubligase. A-C, dominant negative Cul1 constructs block Plk2-dependent loss of SPAR in hippocampal neurons. Dissociated rat hippocampal neurons (DIV16) were transfected with dominant negative Cullin plasmids (B and C) or a control plasmid (A) and super-infected 2 days later with FLAG-tagged Plk2 driven by Sindbis virus (Sin-Plk2). Neurons were fixed ∼18 h post-infection and immunostained for endogenous SPAR and infected Plk2. Transfected cells were identified during image acquisition by the presence of a co-transfected “fill” protein (GFP, seen in the first column of images). SPAR (green) and Plk2 (red) were pseudo-colored for illustrative purposes after image analysis. Arrows point to cells that are both transfected and infected; arrowheads point to cells that are infected only. Yellow indicates the presence of both SPAR and Plk2 staining. D and E, quantification of SPAR immunostaining in somatic and proximal dendritic regions as integrated immunofluorescence intensity per area in cells transfected with indicated plasmids and/or infected with Plk2 Sindbis virus (Sin-Plk2), normalized to nearby untransfected (untr) cells. Values represent the mean ± S.E., n > 17 cells for all conditions, ***, p < 0.001, Mann-Whitney test (E).
Mentions: Cultured hippocampal neurons (16 days in vitro (DIV16)) were transfected with DNCul1 and then super-infected 2 days later with Sindbis virus driving expression of FLAG-tagged Plk2 for ∼18 h to promote degradation of endogenous SPAR (6). We infected at a titer that resulted in ∼10% infection rate of cells already transfected with DNCul1 (Fig. 1, A-C). The option of co-transfecting plasmids driving Plk2 expression was precluded by the low Plk2 expression achievable using this method.6

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