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Protein kinase CK2 contributes to the organization of sodium channels in axonal membranes by regulating their interactions with ankyrin G.

Bréchet A, Fache MP, Brachet A, Ferracci G, Baude A, Irondelle M, Pereira S, Leterrier C, Dargent B - J. Cell Biol. (2008)

Bottom Line: We found that the ankyrin-binding motif of Na(v)1.2 that determines channel concentration at the AIS depends on a glutamate residue (E1111), but also on several serine residues (S1112, S1124, and S1126).Finally, inhibition of CK2 activity reduced sodium channel accumulation at the AIS of neurons.In conclusion, CK2 contributes to sodium channel organization by regulating their interaction with ankyrin G.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 641, Marseille F-13916, France.

ABSTRACT
In neurons, generation and propagation of action potentials requires the precise accumulation of sodium channels at the axonal initial segment (AIS) and in the nodes of Ranvier through ankyrin G scaffolding. We found that the ankyrin-binding motif of Na(v)1.2 that determines channel concentration at the AIS depends on a glutamate residue (E1111), but also on several serine residues (S1112, S1124, and S1126). We showed that phosphorylation of these residues by protein kinase CK2 (CK2) regulates Na(v) channel interaction with ankyrins. Furthermore, we observed that CK2 is highly enriched at the AIS and the nodes of Ranvier in vivo. An ion channel chimera containing the Na(v)1.2 ankyrin-binding motif perturbed endogenous sodium channel accumulation at the AIS, whereas phosphorylation-deficient chimeras did not. Finally, inhibition of CK2 activity reduced sodium channel accumulation at the AIS of neurons. In conclusion, CK2 contributes to sodium channel organization by regulating their interaction with ankyrin G.

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CK2 phosphorylates the Nav1.2 ankyrin-binding motif and regulates its interaction with MBD-ank. (A–C) The Nav1.2 ankyrin-binding motif is phosphorylated by CK2 in vitro. (A) Schematic representation of GST-Nav1.2 linker II-III constructs. The position of the ankyrin-binding motif is indicated by gray boxes. Numbers refer to the position of corresponding amino acid residue in Nav1.2. (B) The constructs were subjected to in vitro CK2 phosphorylation followed by SDS-PAGE. Coomassie brilliant blue staining (CBB) and 32P incorporation revealed by autoradiography are shown. (C) The effect of site-directed serine mutations on CK2 phosphorylation of GST-Nav1.2 II-III. CBB staining (bottom) and 32P incorporation revealed by autoradiography (top) are shown. Numbers to the right of the gel blots indicate the molecular mass of standard markers (in kD). (D–F) SPR analysis of the interaction between the Nav1.2 ankyrin-binding motif and MBD-ank. Typical sensorgrams are illustrated. Increasing concentrations of MBD-ankB ranging from 0.1 to 250 nM were injected over immobilized GST-Nav1.2 1,080–1,203 (D), GST-Nav1.2 989–1,133 (E), and GST-Nav1.2 1,080–1,203 E1111A (F) before (top) and after in situ CK2 phosphorylation (bottom) of the immobilized constructs.
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fig3: CK2 phosphorylates the Nav1.2 ankyrin-binding motif and regulates its interaction with MBD-ank. (A–C) The Nav1.2 ankyrin-binding motif is phosphorylated by CK2 in vitro. (A) Schematic representation of GST-Nav1.2 linker II-III constructs. The position of the ankyrin-binding motif is indicated by gray boxes. Numbers refer to the position of corresponding amino acid residue in Nav1.2. (B) The constructs were subjected to in vitro CK2 phosphorylation followed by SDS-PAGE. Coomassie brilliant blue staining (CBB) and 32P incorporation revealed by autoradiography are shown. (C) The effect of site-directed serine mutations on CK2 phosphorylation of GST-Nav1.2 II-III. CBB staining (bottom) and 32P incorporation revealed by autoradiography (top) are shown. Numbers to the right of the gel blots indicate the molecular mass of standard markers (in kD). (D–F) SPR analysis of the interaction between the Nav1.2 ankyrin-binding motif and MBD-ank. Typical sensorgrams are illustrated. Increasing concentrations of MBD-ankB ranging from 0.1 to 250 nM were injected over immobilized GST-Nav1.2 1,080–1,203 (D), GST-Nav1.2 989–1,133 (E), and GST-Nav1.2 1,080–1,203 E1111A (F) before (top) and after in situ CK2 phosphorylation (bottom) of the immobilized constructs.

Mentions: The ankyrin-binding motif of sodium channels directly interacts with a highly conserved domain in ankyrin G and B called the MBD (Srinivasan et al., 1992; Lemaillet et al., 2003; Mohler et al., 2004). We next examined the possibility that CK2-mediated phosphorylation regulates this interaction. We first determined whether the ankyrin-binding motif of Nav1.2 is phosphorylated by CK2 by conducting in vitro phosphorylation assays with GST proteins fused either to the linker II-III of Nav1.2 (Nav1.2 II-III) or truncated mutants (Fig. 3 A). In the presence of CK2, Nav1.2 II-III was phosphorylated in vitro, as visualized by autoradiography (Fig. 3 B). A similar result was obtained when the C terminus of the Nav1.2 II-III construct was truncated at residue 1133, whereas the deletion of the AIS motif (GST-Nav1.2 989–1,079) resulted in a loss of signal. A positive signal was observed with a GST protein bearing the segment encompassing the AIS motif (GST-Nav1.2 1,080–1,203). Several site-directed mutations of GST-Nav1.2 1,080–1,203 were generated. The respective abrogation of S1112, S1123, or S1124 did not affect in vitro phosphorylation of GST-Nav1.2 1,080–1,203 by CK2 (Fig. 3 C). In contrast, S1126A mutation and the double mutation S1123-24A resulted in a decrease in signal. The double mutation (S1124-26A) and the triple mutation (S1112-24-26A) impaired GST-Nav1.2 1,080–1,203 phosphorylation (Fig. 3 C). All together, these results indicated that the ankyrin-binding motif of Nav1.2 is phosphorylated in vitro by CK2. We next examined whether CK2 phosphorylation regulates the association of the ankyrin-binding motif and MBD-ankyrin (MBD-ank). With this aim, we used SPR technology (Wilson, 2002; Rich and Myszka, 2007). Purified GST-Nav1.2 1,080–1,203 was immobilized on the sensor surface by immunoaffinity. GST–MBD-ank were purified, and the GST-tag was removed using PreScission Protease (see Materials and methods). When an increasing concentration of MBD-ankB was injected over flow cells, a weak surface reactivity was observed (Fig. 3 D). Binding analysis (see Materials and methods) indicated that these two domains associate with an apparent affinity constant (KD) of 1.2 ± 0.4 10−6 M (Table I). When MBD-ankG was substituted for MBD-ankB, a similar affinity constant was obtained (a KD of 1.7± 0.5 10−6 M; Table I). To evaluate the impact of phosphorylation on the interaction, CK2 phosphorylation was performed on immobilized GST-Nav1.2 1080–1203, before either MBD-ankG or MBD-ankB injections (see Materials and methods). Under these conditions, increasing concentrations of MBD-ank injections produced a strongly increased binding signal. Kinetic analysis (see Materials and methods) of the interaction between MBD-ankG and the CK2-phosphorylated GST-Nav1.2 1,080–1,203 gave an association rate (kon) of 42.4 ± 41.6 105 M−1s−1 and a dissociation rate (koff) of 2.1 ± 0.8 10−3s−1, resulting in a KD of 0.9 ± 0.7 10−9 M. Similar binding affinities were obtained with MBD-ankB (Fig. 3 D and Table I). When GST-Nav1.2 989–1,133 was immobilized on the sensor surface, interaction with MBD-ankB was also strongly modulated by CK2 phosphorylation (Fig. 3 E and Table I). These affinity constants were similar to those obtained with GST-Nav1.2 1,080–1,203 (Table I), which indicates that they are independent of the type of immobilized GST-Nav1.2 II-III constructs. We further examined the impact of Nav1.2 E1111 on the association between sodium channels and ankyrins by SPR. In the absence of phosphorylation, immobilized GST-Nav1.2 1,080–1,203 E1111A failed to recruit MBD-ank (Fig. 3 F). After on-chip CK2 phosphorylation, GST Nav1.2 1,080–1,203 E1111A associated with MBD-ank with an affinity constant (KD) of 2.4 ± 0.8 10−8 M (Fig. 3 F and Table I). Finally, we evaluated the contribution of each of the serine residues of the ankyrin-binding motif on the phospho-dependent association between GST-Nav1.2 1,080–1,203 and MBD-ank. The single serines to alanine mutation and double mutation S1123-24A did not modify the phospho-dependent association (Table II). The double mutation (S1124-26A) led to a decrease in affinity (KD = 1.6 ± 1.3 10−7 M; Table II). The triple mutation robustly altered association, resulting in a 1,000-fold decrease in affinity (KD = 2.6 ± 1.1 10−6 M; Table II). All together, these in vitro observations revealed that the association between the ankyrin-binding motif of neuronal sodium channels and the MBD of axonal ankyrins (MBD-ankG and MBD-ankB) is regulated by CK2 phosphorylation. These findings strongly suggest that CK2 is able to strengthen the interaction between ankyrin and sodium channels.


Protein kinase CK2 contributes to the organization of sodium channels in axonal membranes by regulating their interactions with ankyrin G.

Bréchet A, Fache MP, Brachet A, Ferracci G, Baude A, Irondelle M, Pereira S, Leterrier C, Dargent B - J. Cell Biol. (2008)

CK2 phosphorylates the Nav1.2 ankyrin-binding motif and regulates its interaction with MBD-ank. (A–C) The Nav1.2 ankyrin-binding motif is phosphorylated by CK2 in vitro. (A) Schematic representation of GST-Nav1.2 linker II-III constructs. The position of the ankyrin-binding motif is indicated by gray boxes. Numbers refer to the position of corresponding amino acid residue in Nav1.2. (B) The constructs were subjected to in vitro CK2 phosphorylation followed by SDS-PAGE. Coomassie brilliant blue staining (CBB) and 32P incorporation revealed by autoradiography are shown. (C) The effect of site-directed serine mutations on CK2 phosphorylation of GST-Nav1.2 II-III. CBB staining (bottom) and 32P incorporation revealed by autoradiography (top) are shown. Numbers to the right of the gel blots indicate the molecular mass of standard markers (in kD). (D–F) SPR analysis of the interaction between the Nav1.2 ankyrin-binding motif and MBD-ank. Typical sensorgrams are illustrated. Increasing concentrations of MBD-ankB ranging from 0.1 to 250 nM were injected over immobilized GST-Nav1.2 1,080–1,203 (D), GST-Nav1.2 989–1,133 (E), and GST-Nav1.2 1,080–1,203 E1111A (F) before (top) and after in situ CK2 phosphorylation (bottom) of the immobilized constructs.
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fig3: CK2 phosphorylates the Nav1.2 ankyrin-binding motif and regulates its interaction with MBD-ank. (A–C) The Nav1.2 ankyrin-binding motif is phosphorylated by CK2 in vitro. (A) Schematic representation of GST-Nav1.2 linker II-III constructs. The position of the ankyrin-binding motif is indicated by gray boxes. Numbers refer to the position of corresponding amino acid residue in Nav1.2. (B) The constructs were subjected to in vitro CK2 phosphorylation followed by SDS-PAGE. Coomassie brilliant blue staining (CBB) and 32P incorporation revealed by autoradiography are shown. (C) The effect of site-directed serine mutations on CK2 phosphorylation of GST-Nav1.2 II-III. CBB staining (bottom) and 32P incorporation revealed by autoradiography (top) are shown. Numbers to the right of the gel blots indicate the molecular mass of standard markers (in kD). (D–F) SPR analysis of the interaction between the Nav1.2 ankyrin-binding motif and MBD-ank. Typical sensorgrams are illustrated. Increasing concentrations of MBD-ankB ranging from 0.1 to 250 nM were injected over immobilized GST-Nav1.2 1,080–1,203 (D), GST-Nav1.2 989–1,133 (E), and GST-Nav1.2 1,080–1,203 E1111A (F) before (top) and after in situ CK2 phosphorylation (bottom) of the immobilized constructs.
Mentions: The ankyrin-binding motif of sodium channels directly interacts with a highly conserved domain in ankyrin G and B called the MBD (Srinivasan et al., 1992; Lemaillet et al., 2003; Mohler et al., 2004). We next examined the possibility that CK2-mediated phosphorylation regulates this interaction. We first determined whether the ankyrin-binding motif of Nav1.2 is phosphorylated by CK2 by conducting in vitro phosphorylation assays with GST proteins fused either to the linker II-III of Nav1.2 (Nav1.2 II-III) or truncated mutants (Fig. 3 A). In the presence of CK2, Nav1.2 II-III was phosphorylated in vitro, as visualized by autoradiography (Fig. 3 B). A similar result was obtained when the C terminus of the Nav1.2 II-III construct was truncated at residue 1133, whereas the deletion of the AIS motif (GST-Nav1.2 989–1,079) resulted in a loss of signal. A positive signal was observed with a GST protein bearing the segment encompassing the AIS motif (GST-Nav1.2 1,080–1,203). Several site-directed mutations of GST-Nav1.2 1,080–1,203 were generated. The respective abrogation of S1112, S1123, or S1124 did not affect in vitro phosphorylation of GST-Nav1.2 1,080–1,203 by CK2 (Fig. 3 C). In contrast, S1126A mutation and the double mutation S1123-24A resulted in a decrease in signal. The double mutation (S1124-26A) and the triple mutation (S1112-24-26A) impaired GST-Nav1.2 1,080–1,203 phosphorylation (Fig. 3 C). All together, these results indicated that the ankyrin-binding motif of Nav1.2 is phosphorylated in vitro by CK2. We next examined whether CK2 phosphorylation regulates the association of the ankyrin-binding motif and MBD-ankyrin (MBD-ank). With this aim, we used SPR technology (Wilson, 2002; Rich and Myszka, 2007). Purified GST-Nav1.2 1,080–1,203 was immobilized on the sensor surface by immunoaffinity. GST–MBD-ank were purified, and the GST-tag was removed using PreScission Protease (see Materials and methods). When an increasing concentration of MBD-ankB was injected over flow cells, a weak surface reactivity was observed (Fig. 3 D). Binding analysis (see Materials and methods) indicated that these two domains associate with an apparent affinity constant (KD) of 1.2 ± 0.4 10−6 M (Table I). When MBD-ankG was substituted for MBD-ankB, a similar affinity constant was obtained (a KD of 1.7± 0.5 10−6 M; Table I). To evaluate the impact of phosphorylation on the interaction, CK2 phosphorylation was performed on immobilized GST-Nav1.2 1080–1203, before either MBD-ankG or MBD-ankB injections (see Materials and methods). Under these conditions, increasing concentrations of MBD-ank injections produced a strongly increased binding signal. Kinetic analysis (see Materials and methods) of the interaction between MBD-ankG and the CK2-phosphorylated GST-Nav1.2 1,080–1,203 gave an association rate (kon) of 42.4 ± 41.6 105 M−1s−1 and a dissociation rate (koff) of 2.1 ± 0.8 10−3s−1, resulting in a KD of 0.9 ± 0.7 10−9 M. Similar binding affinities were obtained with MBD-ankB (Fig. 3 D and Table I). When GST-Nav1.2 989–1,133 was immobilized on the sensor surface, interaction with MBD-ankB was also strongly modulated by CK2 phosphorylation (Fig. 3 E and Table I). These affinity constants were similar to those obtained with GST-Nav1.2 1,080–1,203 (Table I), which indicates that they are independent of the type of immobilized GST-Nav1.2 II-III constructs. We further examined the impact of Nav1.2 E1111 on the association between sodium channels and ankyrins by SPR. In the absence of phosphorylation, immobilized GST-Nav1.2 1,080–1,203 E1111A failed to recruit MBD-ank (Fig. 3 F). After on-chip CK2 phosphorylation, GST Nav1.2 1,080–1,203 E1111A associated with MBD-ank with an affinity constant (KD) of 2.4 ± 0.8 10−8 M (Fig. 3 F and Table I). Finally, we evaluated the contribution of each of the serine residues of the ankyrin-binding motif on the phospho-dependent association between GST-Nav1.2 1,080–1,203 and MBD-ank. The single serines to alanine mutation and double mutation S1123-24A did not modify the phospho-dependent association (Table II). The double mutation (S1124-26A) led to a decrease in affinity (KD = 1.6 ± 1.3 10−7 M; Table II). The triple mutation robustly altered association, resulting in a 1,000-fold decrease in affinity (KD = 2.6 ± 1.1 10−6 M; Table II). All together, these in vitro observations revealed that the association between the ankyrin-binding motif of neuronal sodium channels and the MBD of axonal ankyrins (MBD-ankG and MBD-ankB) is regulated by CK2 phosphorylation. These findings strongly suggest that CK2 is able to strengthen the interaction between ankyrin and sodium channels.

Bottom Line: We found that the ankyrin-binding motif of Na(v)1.2 that determines channel concentration at the AIS depends on a glutamate residue (E1111), but also on several serine residues (S1112, S1124, and S1126).Finally, inhibition of CK2 activity reduced sodium channel accumulation at the AIS of neurons.In conclusion, CK2 contributes to sodium channel organization by regulating their interaction with ankyrin G.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 641, Marseille F-13916, France.

ABSTRACT
In neurons, generation and propagation of action potentials requires the precise accumulation of sodium channels at the axonal initial segment (AIS) and in the nodes of Ranvier through ankyrin G scaffolding. We found that the ankyrin-binding motif of Na(v)1.2 that determines channel concentration at the AIS depends on a glutamate residue (E1111), but also on several serine residues (S1112, S1124, and S1126). We showed that phosphorylation of these residues by protein kinase CK2 (CK2) regulates Na(v) channel interaction with ankyrins. Furthermore, we observed that CK2 is highly enriched at the AIS and the nodes of Ranvier in vivo. An ion channel chimera containing the Na(v)1.2 ankyrin-binding motif perturbed endogenous sodium channel accumulation at the AIS, whereas phosphorylation-deficient chimeras did not. Finally, inhibition of CK2 activity reduced sodium channel accumulation at the AIS of neurons. In conclusion, CK2 contributes to sodium channel organization by regulating their interaction with ankyrin G.

Show MeSH
Related in: MedlinePlus