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Mobile DHHC palmitoylating enzyme mediates activity-sensitive synaptic targeting of PSD-95.

Noritake J, Fukata Y, Iwanaga T, Hosomi N, Tsutsumi R, Matsuda N, Tani H, Iwanari H, Mochizuki Y, Kodama T, Matsuura Y, Bredt DS, Hamakubo T, Fukata M - J. Cell Biol. (2009)

Bottom Line: We found that blocking synaptic activity rapidly induces PSD-95 palmitoylation and mediates synaptic clustering of PSD-95 and associated AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors.Upon activity blockade, DHHC2 translocates to the postsynaptic density to transduce this effect.These data demonstrate that individual DHHC members are differentially regulated and that dynamic recruitment of protein palmitoylation machinery enables compartmentalized regulation of protein trafficking in response to extracellular signals.

View Article: PubMed Central - PubMed

Affiliation: Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki, Aichi, Japan.

ABSTRACT
Protein palmitoylation is the most common posttranslational lipid modification; its reversibility mediates protein shuttling between intracellular compartments. A large family of DHHC (Asp-His-His-Cys) proteins has emerged as protein palmitoyl acyltransferases (PATs). However, mechanisms that regulate these PATs in a physiological context remain unknown. In this study, we efficiently monitored the dynamic palmitate cycling on synaptic scaffold PSD-95. We found that blocking synaptic activity rapidly induces PSD-95 palmitoylation and mediates synaptic clustering of PSD-95 and associated AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors. A dendritically localized DHHC2 but not the Golgi-resident DHHC3 mediates this activity-sensitive palmitoylation. Upon activity blockade, DHHC2 translocates to the postsynaptic density to transduce this effect. These data demonstrate that individual DHHC members are differentially regulated and that dynamic recruitment of protein palmitoylation machinery enables compartmentalized regulation of protein trafficking in response to extracellular signals.

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The DHHC2/15 subfamily of PSD-95 PATs is regulated by synaptic activity. (A) Activity blockade induces quantitative palmitoylation of PSD-95 but not Gαq. Hydroxylamine (H)-sensitive palmitoylated proteins were purified from treated neurons by the ABE method. The amount of palmitoylated PSD-95 and Gαq was analyzed by Western blotting. T, Tris treatment as a control of hydroxylamine. (B and C) Kyn-induced PSD-95 palmitoylation and synaptic accumulation were reversible upon washing out Kyn. (B) Treatment of hippocampal neurons with Kyn for 2 h enhanced PSD-95 palmitoylation. After washout, PSD-95 palmitoylation level returned to the basal level within 2 h (ABE), with consistent mobility change of PSD-95 (−βME). In contrast, Gαq, GluR2, and GRIP1 palmitoylation did not change upon activity blockade. Kyn-induced palmitoylation changes were quantified. n = 3 each; ***, P < 0.001. Error bars indicate SD. The dashed line (100%) indicates the normalized control level. IB, immunoblot. (A and B) Closed and open arrows indicate the positions of palmitoylated and nonpalmitoylated PSD-95, respectively. (C) The increased accumulation of PSD-95–GFP upon Kyn treatment returned to the basal level at 2 h after Kyn washout. (D) Cultured hippocampal neurons expressing a DN mutant of the DHHC2 and -15 subfamily (DN-DH2/15) were treated with 3 mCi/ml [3H]palmitate for 2 h in the presence or absence of Kyn. Immunoprecipitated PSD-95 was resolved by SDS-PAGE, followed by fluorography ([3H]palm) and Coomassie staining (CBB). Inhibition of glutamate receptor activity with Kyn greatly enhanced PSD-95 palmitoylation. This enhancement was decreased by DN-DH2/15. IP, immunoprecipitation. Bar, 5 µm.
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fig2: The DHHC2/15 subfamily of PSD-95 PATs is regulated by synaptic activity. (A) Activity blockade induces quantitative palmitoylation of PSD-95 but not Gαq. Hydroxylamine (H)-sensitive palmitoylated proteins were purified from treated neurons by the ABE method. The amount of palmitoylated PSD-95 and Gαq was analyzed by Western blotting. T, Tris treatment as a control of hydroxylamine. (B and C) Kyn-induced PSD-95 palmitoylation and synaptic accumulation were reversible upon washing out Kyn. (B) Treatment of hippocampal neurons with Kyn for 2 h enhanced PSD-95 palmitoylation. After washout, PSD-95 palmitoylation level returned to the basal level within 2 h (ABE), with consistent mobility change of PSD-95 (−βME). In contrast, Gαq, GluR2, and GRIP1 palmitoylation did not change upon activity blockade. Kyn-induced palmitoylation changes were quantified. n = 3 each; ***, P < 0.001. Error bars indicate SD. The dashed line (100%) indicates the normalized control level. IB, immunoblot. (A and B) Closed and open arrows indicate the positions of palmitoylated and nonpalmitoylated PSD-95, respectively. (C) The increased accumulation of PSD-95–GFP upon Kyn treatment returned to the basal level at 2 h after Kyn washout. (D) Cultured hippocampal neurons expressing a DN mutant of the DHHC2 and -15 subfamily (DN-DH2/15) were treated with 3 mCi/ml [3H]palmitate for 2 h in the presence or absence of Kyn. Immunoprecipitated PSD-95 was resolved by SDS-PAGE, followed by fluorography ([3H]palm) and Coomassie staining (CBB). Inhibition of glutamate receptor activity with Kyn greatly enhanced PSD-95 palmitoylation. This enhancement was decreased by DN-DH2/15. IP, immunoprecipitation. Bar, 5 µm.

Mentions: To monitor PSD-95 palmitoylation biochemically, we used the acyl-biotin exchange (ABE) method (Roth et al., 2006; Kang et al., 2008). We confirmed that this method specifically identified palmitoylated proteins, including PSD-95, in heterologous cells (Fig. S2 A). As previously reported (El-Husseini et al., 2002), treating neurons for 12 h with 2-BP reduced PSD-95 palmitoylation (palmitoylated PSD-95 = 13 ± 15% of control cells; P < 0.001; Fig. 2 A). When we treated neurons for 2 h with Kyn, the amount of palmitoylated PSD-95 significantly increased (198 ± 13% of control cells; P < 0.001; Fig. 2, A and B). Blocking glutamate receptors with a combination of APV (D-[-]-2-amino-5-phosphonopentanoic acid), which blocks NMDA receptors, and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), which blocks AMPARs, also enhanced PSD-95 palmitoylation within 2 h (palmitoylated PSD-95 = 184 ± 23% of control cells; P < 0.01). 2-BP blocked the rapid enhancement of PSD-95 palmitoylation, indicating that inhibition of depalmitoylation is not solely responsible and that newly occurring palmitoylation mediates this effect. This activity-sensitive PSD-95 palmitoylation is stoichiometric, as Kyn and APV + CNQX quantitatively shifted the PSD-95 band upward (Fig. 2, A and B; Fig. S1 C; and Fig. S2 B). This upward shift reflects palmitoylation, as β-mercaptoethanol (βME), which hydrolyses the palmitoyl thioester, leaves only the lower band (Fig. 2, A and B, bottom). Both the increased PSD-95 palmitoylation and synaptic accumulation were reversible upon washing out of Kyn, indicating that this process is activity sensitive (Fig. 2, B and C).


Mobile DHHC palmitoylating enzyme mediates activity-sensitive synaptic targeting of PSD-95.

Noritake J, Fukata Y, Iwanaga T, Hosomi N, Tsutsumi R, Matsuda N, Tani H, Iwanari H, Mochizuki Y, Kodama T, Matsuura Y, Bredt DS, Hamakubo T, Fukata M - J. Cell Biol. (2009)

The DHHC2/15 subfamily of PSD-95 PATs is regulated by synaptic activity. (A) Activity blockade induces quantitative palmitoylation of PSD-95 but not Gαq. Hydroxylamine (H)-sensitive palmitoylated proteins were purified from treated neurons by the ABE method. The amount of palmitoylated PSD-95 and Gαq was analyzed by Western blotting. T, Tris treatment as a control of hydroxylamine. (B and C) Kyn-induced PSD-95 palmitoylation and synaptic accumulation were reversible upon washing out Kyn. (B) Treatment of hippocampal neurons with Kyn for 2 h enhanced PSD-95 palmitoylation. After washout, PSD-95 palmitoylation level returned to the basal level within 2 h (ABE), with consistent mobility change of PSD-95 (−βME). In contrast, Gαq, GluR2, and GRIP1 palmitoylation did not change upon activity blockade. Kyn-induced palmitoylation changes were quantified. n = 3 each; ***, P < 0.001. Error bars indicate SD. The dashed line (100%) indicates the normalized control level. IB, immunoblot. (A and B) Closed and open arrows indicate the positions of palmitoylated and nonpalmitoylated PSD-95, respectively. (C) The increased accumulation of PSD-95–GFP upon Kyn treatment returned to the basal level at 2 h after Kyn washout. (D) Cultured hippocampal neurons expressing a DN mutant of the DHHC2 and -15 subfamily (DN-DH2/15) were treated with 3 mCi/ml [3H]palmitate for 2 h in the presence or absence of Kyn. Immunoprecipitated PSD-95 was resolved by SDS-PAGE, followed by fluorography ([3H]palm) and Coomassie staining (CBB). Inhibition of glutamate receptor activity with Kyn greatly enhanced PSD-95 palmitoylation. This enhancement was decreased by DN-DH2/15. IP, immunoprecipitation. Bar, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2712995&req=5

fig2: The DHHC2/15 subfamily of PSD-95 PATs is regulated by synaptic activity. (A) Activity blockade induces quantitative palmitoylation of PSD-95 but not Gαq. Hydroxylamine (H)-sensitive palmitoylated proteins were purified from treated neurons by the ABE method. The amount of palmitoylated PSD-95 and Gαq was analyzed by Western blotting. T, Tris treatment as a control of hydroxylamine. (B and C) Kyn-induced PSD-95 palmitoylation and synaptic accumulation were reversible upon washing out Kyn. (B) Treatment of hippocampal neurons with Kyn for 2 h enhanced PSD-95 palmitoylation. After washout, PSD-95 palmitoylation level returned to the basal level within 2 h (ABE), with consistent mobility change of PSD-95 (−βME). In contrast, Gαq, GluR2, and GRIP1 palmitoylation did not change upon activity blockade. Kyn-induced palmitoylation changes were quantified. n = 3 each; ***, P < 0.001. Error bars indicate SD. The dashed line (100%) indicates the normalized control level. IB, immunoblot. (A and B) Closed and open arrows indicate the positions of palmitoylated and nonpalmitoylated PSD-95, respectively. (C) The increased accumulation of PSD-95–GFP upon Kyn treatment returned to the basal level at 2 h after Kyn washout. (D) Cultured hippocampal neurons expressing a DN mutant of the DHHC2 and -15 subfamily (DN-DH2/15) were treated with 3 mCi/ml [3H]palmitate for 2 h in the presence or absence of Kyn. Immunoprecipitated PSD-95 was resolved by SDS-PAGE, followed by fluorography ([3H]palm) and Coomassie staining (CBB). Inhibition of glutamate receptor activity with Kyn greatly enhanced PSD-95 palmitoylation. This enhancement was decreased by DN-DH2/15. IP, immunoprecipitation. Bar, 5 µm.
Mentions: To monitor PSD-95 palmitoylation biochemically, we used the acyl-biotin exchange (ABE) method (Roth et al., 2006; Kang et al., 2008). We confirmed that this method specifically identified palmitoylated proteins, including PSD-95, in heterologous cells (Fig. S2 A). As previously reported (El-Husseini et al., 2002), treating neurons for 12 h with 2-BP reduced PSD-95 palmitoylation (palmitoylated PSD-95 = 13 ± 15% of control cells; P < 0.001; Fig. 2 A). When we treated neurons for 2 h with Kyn, the amount of palmitoylated PSD-95 significantly increased (198 ± 13% of control cells; P < 0.001; Fig. 2, A and B). Blocking glutamate receptors with a combination of APV (D-[-]-2-amino-5-phosphonopentanoic acid), which blocks NMDA receptors, and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), which blocks AMPARs, also enhanced PSD-95 palmitoylation within 2 h (palmitoylated PSD-95 = 184 ± 23% of control cells; P < 0.01). 2-BP blocked the rapid enhancement of PSD-95 palmitoylation, indicating that inhibition of depalmitoylation is not solely responsible and that newly occurring palmitoylation mediates this effect. This activity-sensitive PSD-95 palmitoylation is stoichiometric, as Kyn and APV + CNQX quantitatively shifted the PSD-95 band upward (Fig. 2, A and B; Fig. S1 C; and Fig. S2 B). This upward shift reflects palmitoylation, as β-mercaptoethanol (βME), which hydrolyses the palmitoyl thioester, leaves only the lower band (Fig. 2, A and B, bottom). Both the increased PSD-95 palmitoylation and synaptic accumulation were reversible upon washing out of Kyn, indicating that this process is activity sensitive (Fig. 2, B and C).

Bottom Line: We found that blocking synaptic activity rapidly induces PSD-95 palmitoylation and mediates synaptic clustering of PSD-95 and associated AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors.Upon activity blockade, DHHC2 translocates to the postsynaptic density to transduce this effect.These data demonstrate that individual DHHC members are differentially regulated and that dynamic recruitment of protein palmitoylation machinery enables compartmentalized regulation of protein trafficking in response to extracellular signals.

View Article: PubMed Central - PubMed

Affiliation: Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki, Aichi, Japan.

ABSTRACT
Protein palmitoylation is the most common posttranslational lipid modification; its reversibility mediates protein shuttling between intracellular compartments. A large family of DHHC (Asp-His-His-Cys) proteins has emerged as protein palmitoyl acyltransferases (PATs). However, mechanisms that regulate these PATs in a physiological context remain unknown. In this study, we efficiently monitored the dynamic palmitate cycling on synaptic scaffold PSD-95. We found that blocking synaptic activity rapidly induces PSD-95 palmitoylation and mediates synaptic clustering of PSD-95 and associated AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors. A dendritically localized DHHC2 but not the Golgi-resident DHHC3 mediates this activity-sensitive palmitoylation. Upon activity blockade, DHHC2 translocates to the postsynaptic density to transduce this effect. These data demonstrate that individual DHHC members are differentially regulated and that dynamic recruitment of protein palmitoylation machinery enables compartmentalized regulation of protein trafficking in response to extracellular signals.

Show MeSH
Related in: MedlinePlus