Limits...
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.

Show MeSH

Related in: MedlinePlus

A candidateβ-TRCP phosphodegron in SPAR. A, schematic representation of SPAR domains and SPAR fragments: actin-binding domains (Act1 and Act2), RapGAP, PDZ, and guanylate kinase binding (GKBD) domain. The candidate DSGIDT phosphodegron motif (residues 1304-1309) identified in the Act2 domain of SPAR closely resembles the consensus β-TRCP phosphodegron (inset). Boundaries of generated C-terminal fragments of SPAR are depicted; C-1, C-2, and Act2 fragments contain the putative phosphodegron motif, whereas C-3 does not. B and C, SPAR fragments spanning the β-TRCP phosphodegron bind to β-TRCP. HEK293T cells were transfected with vectors expressing SPAR fragments C-1, C-2, C-3 (B) or Act2 (C) (0.6 μg) either alone or with pCMV-GST or pCMV-GST-β-TRCP (0.6 μg). In addition, pCMV-HA-Plk2WT/K108M (0.6 μg) and DNCul1 (2 μg) were co-transfected as indicated. After 24 h, cell extracts were used for GSH-Sepharose pulldown assays, and proteins were immunoblotted with Myc antibodies. Crude lysates were blotted as an input control. D and E, phosphodegron-dependent binding to β-TRCP. Constructs expressing point mutations (S1305A, T1309A) in full-length SPAR (E) and the Act2 fragment (D) (myc-SPARAA or myc-Act2AA, 0.6 μg) were transfected into HEK293T cells along with pCMV-HA-Plk2WT/D201A (0.6 μg), pCMV-DNCul1 (2 μg), and pCMV-GST or pCMV-GST-β-TRCP (0.6 μg) as indicated. Cell lysates were incubated with GSH-Sepharose and immunoblotted with Myc antibodies. Crude extracts were resolved to control for input. Wild type constructs expressing myc-SPAR and myc-Act2 were used for comparison as a positive control for interaction with GST-β-TRCP. F, SPAR associates with endogenous SCFβ-TRCP1 complex in the presence of active Plk2 and dependent upon its phosphodegron. Constructs expressing full-length myc-SPAR (WT or AA) were co-expressed in HEK293T cells with active (WT) or catalytically inactive Plk2 (D201A). Before lysis and immunoprecipitation with 9E10-agarose, cells were treated with proteasome inhibitor MG-132 (25 μm) for 5 h. Proteins bound to 9E10-agarose were analyzed via immunoblotting using Myc antibodies and antibodies that recognized endogenous β-TRCP1 and Cul1. Myc-SPAR-C3, a fragment of SPAR that does not contain the phosphodegron served as a negative control, and myc-Cdc25A, a known target of SCFβ-TRCP1, served as a positive control. G, phosphodegron-dependent degradation of SPAR by Plk2 and β-TRCP. Myc-SPARWT and myc-SPARAA abundance was compared by immunoblotting with the indicated antibodies in HEK293T cells in the absence and presence of pCMV-HA-Plk2 in the background of empty vector or β-TRCPΔF.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2570879&req=5

fig4: A candidateβ-TRCP phosphodegron in SPAR. A, schematic representation of SPAR domains and SPAR fragments: actin-binding domains (Act1 and Act2), RapGAP, PDZ, and guanylate kinase binding (GKBD) domain. The candidate DSGIDT phosphodegron motif (residues 1304-1309) identified in the Act2 domain of SPAR closely resembles the consensus β-TRCP phosphodegron (inset). Boundaries of generated C-terminal fragments of SPAR are depicted; C-1, C-2, and Act2 fragments contain the putative phosphodegron motif, whereas C-3 does not. B and C, SPAR fragments spanning the β-TRCP phosphodegron bind to β-TRCP. HEK293T cells were transfected with vectors expressing SPAR fragments C-1, C-2, C-3 (B) or Act2 (C) (0.6 μg) either alone or with pCMV-GST or pCMV-GST-β-TRCP (0.6 μg). In addition, pCMV-HA-Plk2WT/K108M (0.6 μg) and DNCul1 (2 μg) were co-transfected as indicated. After 24 h, cell extracts were used for GSH-Sepharose pulldown assays, and proteins were immunoblotted with Myc antibodies. Crude lysates were blotted as an input control. D and E, phosphodegron-dependent binding to β-TRCP. Constructs expressing point mutations (S1305A, T1309A) in full-length SPAR (E) and the Act2 fragment (D) (myc-SPARAA or myc-Act2AA, 0.6 μg) were transfected into HEK293T cells along with pCMV-HA-Plk2WT/D201A (0.6 μg), pCMV-DNCul1 (2 μg), and pCMV-GST or pCMV-GST-β-TRCP (0.6 μg) as indicated. Cell lysates were incubated with GSH-Sepharose and immunoblotted with Myc antibodies. Crude extracts were resolved to control for input. Wild type constructs expressing myc-SPAR and myc-Act2 were used for comparison as a positive control for interaction with GST-β-TRCP. F, SPAR associates with endogenous SCFβ-TRCP1 complex in the presence of active Plk2 and dependent upon its phosphodegron. Constructs expressing full-length myc-SPAR (WT or AA) were co-expressed in HEK293T cells with active (WT) or catalytically inactive Plk2 (D201A). Before lysis and immunoprecipitation with 9E10-agarose, cells were treated with proteasome inhibitor MG-132 (25 μm) for 5 h. Proteins bound to 9E10-agarose were analyzed via immunoblotting using Myc antibodies and antibodies that recognized endogenous β-TRCP1 and Cul1. Myc-SPAR-C3, a fragment of SPAR that does not contain the phosphodegron served as a negative control, and myc-Cdc25A, a known target of SCFβ-TRCP1, served as a positive control. G, phosphodegron-dependent degradation of SPAR by Plk2 and β-TRCP. Myc-SPARWT and myc-SPARAA abundance was compared by immunoblotting with the indicated antibodies in HEK293T cells in the absence and presence of pCMV-HA-Plk2 in the background of empty vector or β-TRCPΔF.

Mentions: Plk2-dependent Recognition of SPAR by β-TRCP Involves a Canonical Phosphodegron—SPAR is a large protein of 1804 amino acids containing two actin binding domains (Act1 and Act2), a RapGAP domain, a PDZ domain, and a guanylate kinase binding domain (GKBD) (Fig. 4A). Within the Act2 domain, we identified a candidate β-TRCP recognition motif (DSGIDT, residues 1304-1309) based upon the consensus β-TRCP recognition motif found in many of its targets (DpSGΦX(pS/T); Φ= hydrophobic residue, X = any residue, pS or pS/T = phosphoserine or threonine) (Fig. 4A). Initially, we surveyed three fragments of SPAR spanning the C terminus (C-1, C-2, C-3, Fig. 4A) for their ability to interact with co-transfected GST-β-TRCP1 in HEK293T cells in the presence of Plk2 and DNCul1. Fragments C-1 and C-2 (which contain the candidate phosphodegron) bound to GST-β-TRCP, whereas the C-3 construct lacking the phosphodegron failed to do so (Fig. 4B). N-terminal SPAR fragments that lack the DSGIDT motif also could not bind to β-TRCP (data not shown).


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)

A candidateβ-TRCP phosphodegron in SPAR. A, schematic representation of SPAR domains and SPAR fragments: actin-binding domains (Act1 and Act2), RapGAP, PDZ, and guanylate kinase binding (GKBD) domain. The candidate DSGIDT phosphodegron motif (residues 1304-1309) identified in the Act2 domain of SPAR closely resembles the consensus β-TRCP phosphodegron (inset). Boundaries of generated C-terminal fragments of SPAR are depicted; C-1, C-2, and Act2 fragments contain the putative phosphodegron motif, whereas C-3 does not. B and C, SPAR fragments spanning the β-TRCP phosphodegron bind to β-TRCP. HEK293T cells were transfected with vectors expressing SPAR fragments C-1, C-2, C-3 (B) or Act2 (C) (0.6 μg) either alone or with pCMV-GST or pCMV-GST-β-TRCP (0.6 μg). In addition, pCMV-HA-Plk2WT/K108M (0.6 μg) and DNCul1 (2 μg) were co-transfected as indicated. After 24 h, cell extracts were used for GSH-Sepharose pulldown assays, and proteins were immunoblotted with Myc antibodies. Crude lysates were blotted as an input control. D and E, phosphodegron-dependent binding to β-TRCP. Constructs expressing point mutations (S1305A, T1309A) in full-length SPAR (E) and the Act2 fragment (D) (myc-SPARAA or myc-Act2AA, 0.6 μg) were transfected into HEK293T cells along with pCMV-HA-Plk2WT/D201A (0.6 μg), pCMV-DNCul1 (2 μg), and pCMV-GST or pCMV-GST-β-TRCP (0.6 μg) as indicated. Cell lysates were incubated with GSH-Sepharose and immunoblotted with Myc antibodies. Crude extracts were resolved to control for input. Wild type constructs expressing myc-SPAR and myc-Act2 were used for comparison as a positive control for interaction with GST-β-TRCP. F, SPAR associates with endogenous SCFβ-TRCP1 complex in the presence of active Plk2 and dependent upon its phosphodegron. Constructs expressing full-length myc-SPAR (WT or AA) were co-expressed in HEK293T cells with active (WT) or catalytically inactive Plk2 (D201A). Before lysis and immunoprecipitation with 9E10-agarose, cells were treated with proteasome inhibitor MG-132 (25 μm) for 5 h. Proteins bound to 9E10-agarose were analyzed via immunoblotting using Myc antibodies and antibodies that recognized endogenous β-TRCP1 and Cul1. Myc-SPAR-C3, a fragment of SPAR that does not contain the phosphodegron served as a negative control, and myc-Cdc25A, a known target of SCFβ-TRCP1, served as a positive control. G, phosphodegron-dependent degradation of SPAR by Plk2 and β-TRCP. Myc-SPARWT and myc-SPARAA abundance was compared by immunoblotting with the indicated antibodies in HEK293T cells in the absence and presence of pCMV-HA-Plk2 in the background of empty vector or β-TRCPΔF.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: A candidateβ-TRCP phosphodegron in SPAR. A, schematic representation of SPAR domains and SPAR fragments: actin-binding domains (Act1 and Act2), RapGAP, PDZ, and guanylate kinase binding (GKBD) domain. The candidate DSGIDT phosphodegron motif (residues 1304-1309) identified in the Act2 domain of SPAR closely resembles the consensus β-TRCP phosphodegron (inset). Boundaries of generated C-terminal fragments of SPAR are depicted; C-1, C-2, and Act2 fragments contain the putative phosphodegron motif, whereas C-3 does not. B and C, SPAR fragments spanning the β-TRCP phosphodegron bind to β-TRCP. HEK293T cells were transfected with vectors expressing SPAR fragments C-1, C-2, C-3 (B) or Act2 (C) (0.6 μg) either alone or with pCMV-GST or pCMV-GST-β-TRCP (0.6 μg). In addition, pCMV-HA-Plk2WT/K108M (0.6 μg) and DNCul1 (2 μg) were co-transfected as indicated. After 24 h, cell extracts were used for GSH-Sepharose pulldown assays, and proteins were immunoblotted with Myc antibodies. Crude lysates were blotted as an input control. D and E, phosphodegron-dependent binding to β-TRCP. Constructs expressing point mutations (S1305A, T1309A) in full-length SPAR (E) and the Act2 fragment (D) (myc-SPARAA or myc-Act2AA, 0.6 μg) were transfected into HEK293T cells along with pCMV-HA-Plk2WT/D201A (0.6 μg), pCMV-DNCul1 (2 μg), and pCMV-GST or pCMV-GST-β-TRCP (0.6 μg) as indicated. Cell lysates were incubated with GSH-Sepharose and immunoblotted with Myc antibodies. Crude extracts were resolved to control for input. Wild type constructs expressing myc-SPAR and myc-Act2 were used for comparison as a positive control for interaction with GST-β-TRCP. F, SPAR associates with endogenous SCFβ-TRCP1 complex in the presence of active Plk2 and dependent upon its phosphodegron. Constructs expressing full-length myc-SPAR (WT or AA) were co-expressed in HEK293T cells with active (WT) or catalytically inactive Plk2 (D201A). Before lysis and immunoprecipitation with 9E10-agarose, cells were treated with proteasome inhibitor MG-132 (25 μm) for 5 h. Proteins bound to 9E10-agarose were analyzed via immunoblotting using Myc antibodies and antibodies that recognized endogenous β-TRCP1 and Cul1. Myc-SPAR-C3, a fragment of SPAR that does not contain the phosphodegron served as a negative control, and myc-Cdc25A, a known target of SCFβ-TRCP1, served as a positive control. G, phosphodegron-dependent degradation of SPAR by Plk2 and β-TRCP. Myc-SPARWT and myc-SPARAA abundance was compared by immunoblotting with the indicated antibodies in HEK293T cells in the absence and presence of pCMV-HA-Plk2 in the background of empty vector or β-TRCPΔF.
Mentions: Plk2-dependent Recognition of SPAR by β-TRCP Involves a Canonical Phosphodegron—SPAR is a large protein of 1804 amino acids containing two actin binding domains (Act1 and Act2), a RapGAP domain, a PDZ domain, and a guanylate kinase binding domain (GKBD) (Fig. 4A). Within the Act2 domain, we identified a candidate β-TRCP recognition motif (DSGIDT, residues 1304-1309) based upon the consensus β-TRCP recognition motif found in many of its targets (DpSGΦX(pS/T); Φ= hydrophobic residue, X = any residue, pS or pS/T = phosphoserine or threonine) (Fig. 4A). Initially, we surveyed three fragments of SPAR spanning the C terminus (C-1, C-2, C-3, Fig. 4A) for their ability to interact with co-transfected GST-β-TRCP1 in HEK293T cells in the presence of Plk2 and DNCul1. Fragments C-1 and C-2 (which contain the candidate phosphodegron) bound to GST-β-TRCP, whereas the C-3 construct lacking the phosphodegron failed to do so (Fig. 4B). N-terminal SPAR fragments that lack the DSGIDT motif also could not bind to β-TRCP (data not shown).

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