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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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Regulation of SPAR degradation in hippocampal neurons through the SCFβ-TRCP complex. A-C, dominant negative β-TRCP constructs block Plk2-dependent loss of SPAR in hippocampal neurons. As in Fig. 1, DIV16 dissociated rat hippocampal neurons were transfected with dominant negative β-TRCP plasmids (A and B) or control SkpΔF plasmid (C) and superinfected 2 days later with FLAG-tagged Plk2 driven by Sindbis virus (Sin-Plk2). 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 in Fig. 1. Values represent the mean ± S.E. n = 25-37 cells for all constructs in panel D; *, p < 0.05 indicates significant difference from theoretical mean of 100%, Student's t test (D). For panel E, n = 19-37 cells (infected only), n = 13-28 cells (infected and transfected), ***, p < 0.001, Mann Whitney test.
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fig5: Regulation of SPAR degradation in hippocampal neurons through the SCFβ-TRCP complex. A-C, dominant negative β-TRCP constructs block Plk2-dependent loss of SPAR in hippocampal neurons. As in Fig. 1, DIV16 dissociated rat hippocampal neurons were transfected with dominant negative β-TRCP plasmids (A and B) or control SkpΔF plasmid (C) and superinfected 2 days later with FLAG-tagged Plk2 driven by Sindbis virus (Sin-Plk2). 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 in Fig. 1. Values represent the mean ± S.E. n = 25-37 cells for all constructs in panel D; *, p < 0.05 indicates significant difference from theoretical mean of 100%, Student's t test (D). For panel E, n = 19-37 cells (infected only), n = 13-28 cells (infected and transfected), ***, p < 0.001, Mann Whitney test.

Mentions: Does the SCFβ-TRCP complex regulate turnover of SPAR in neurons? Unlike for the human mRNAs, we have yet to identify an shRNA sequence that can target both β-TRCP1 and β-TRCP2 transcripts efficiently in rat neurons. Co-transfection of different shRNAs that individually target β-TRCP1 and β-TRCP2 has not yielded satisfactory knockdown of both proteins, possibly due to low efficiency of cotransfection of both shRNA vectors in the same neuron. Therefore, we opted to disrupt endogenous SCFβ-TRCP function by overexpression of dominant-negative β-TRCP constructs β-TRCP1ΔF or β-TRCP2ΔF. As in Fig. 1, DIV16 cultured hippocampal neurons were transfected with either β-TRCP1ΔFor β-TRCP2ΔF and then super-infected 2 days later with Sindbis virus to drive expression of FLAG-tagged Plk2 and induce SPAR degradation (Fig. 5).


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)

Regulation of SPAR degradation in hippocampal neurons through the SCFβ-TRCP complex. A-C, dominant negative β-TRCP constructs block Plk2-dependent loss of SPAR in hippocampal neurons. As in Fig. 1, DIV16 dissociated rat hippocampal neurons were transfected with dominant negative β-TRCP plasmids (A and B) or control SkpΔF plasmid (C) and superinfected 2 days later with FLAG-tagged Plk2 driven by Sindbis virus (Sin-Plk2). 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 in Fig. 1. Values represent the mean ± S.E. n = 25-37 cells for all constructs in panel D; *, p < 0.05 indicates significant difference from theoretical mean of 100%, Student's t test (D). For panel E, n = 19-37 cells (infected only), n = 13-28 cells (infected and transfected), ***, p < 0.001, Mann Whitney test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Regulation of SPAR degradation in hippocampal neurons through the SCFβ-TRCP complex. A-C, dominant negative β-TRCP constructs block Plk2-dependent loss of SPAR in hippocampal neurons. As in Fig. 1, DIV16 dissociated rat hippocampal neurons were transfected with dominant negative β-TRCP plasmids (A and B) or control SkpΔF plasmid (C) and superinfected 2 days later with FLAG-tagged Plk2 driven by Sindbis virus (Sin-Plk2). 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 in Fig. 1. Values represent the mean ± S.E. n = 25-37 cells for all constructs in panel D; *, p < 0.05 indicates significant difference from theoretical mean of 100%, Student's t test (D). For panel E, n = 19-37 cells (infected only), n = 13-28 cells (infected and transfected), ***, p < 0.001, Mann Whitney test.
Mentions: Does the SCFβ-TRCP complex regulate turnover of SPAR in neurons? Unlike for the human mRNAs, we have yet to identify an shRNA sequence that can target both β-TRCP1 and β-TRCP2 transcripts efficiently in rat neurons. Co-transfection of different shRNAs that individually target β-TRCP1 and β-TRCP2 has not yielded satisfactory knockdown of both proteins, possibly due to low efficiency of cotransfection of both shRNA vectors in the same neuron. Therefore, we opted to disrupt endogenous SCFβ-TRCP function by overexpression of dominant-negative β-TRCP constructs β-TRCP1ΔF or β-TRCP2ΔF. As in Fig. 1, DIV16 cultured hippocampal neurons were transfected with either β-TRCP1ΔFor β-TRCP2ΔF and then super-infected 2 days later with Sindbis virus to drive expression of FLAG-tagged Plk2 and induce SPAR degradation (Fig. 5).

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