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Regulation of Pro-Apoptotic Phosphorylation of Kv2.1 K+ Channels.

He K, McCord MC, Hartnett KA, Aizenman E - PLoS ONE (2015)

Bottom Line: Using immunoprecipitated Kv2.1 protein and phospho-specific antibodies, we found that an intact Y124 is required for p38 phosphorylation of S800, and, importantly, that Src phosphorylation of Y124 facilitates the action of the p38 at the S800 residue.Moreover, the actions of Src on Kv2.1 are substantially decreased in the non-phosphorylatable S800A channel mutant.We also observed that mutations of either C73 or C710 residues decreased the p38 phosphorylation at S800 without influencing the actions of Src on tyrosine phosphorylation of Kv2.1.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Pittsburgh School of Medicine, E1456 BST, 3500 Terrace St., Pittsburgh, PA, 15261, United States of America.

ABSTRACT
Caspase activity during apoptosis is inhibited by physiological concentrations of intracellular K+. To enable apoptosis in injured cortical and hippocampal neurons, cellular loss of this cation is facilitated by the insertion of Kv2.1 K+ channels into the plasma membrane via a Zn2+/CaMKII/SNARE-dependent process. Pro-apoptotic membrane insertion of Kv2.1 requires the dual phosphorylation of the channel by Src and p38 at cytoplasmic N- and C-terminal residues Y124 and S800, respectively. In this study, we investigate if these phosphorylation sites are mutually co-regulated, and whether putative N- and C-terminal interactions, possibly enabled by Kv2.1 intracellular cysteine residues C73 and C710, influence the phosphorylation process itself. Studies were performed with recombinant wild type and mutant Kv2.1 expressed in Chinese hamster ovary (CHO) cells. Using immunoprecipitated Kv2.1 protein and phospho-specific antibodies, we found that an intact Y124 is required for p38 phosphorylation of S800, and, importantly, that Src phosphorylation of Y124 facilitates the action of the p38 at the S800 residue. Moreover, the actions of Src on Kv2.1 are substantially decreased in the non-phosphorylatable S800A channel mutant. We also observed that mutations of either C73 or C710 residues decreased the p38 phosphorylation at S800 without influencing the actions of Src on tyrosine phosphorylation of Kv2.1. Surprisingly, however, apoptotic K+ currents were suppressed only in cells expressing the Kv2.1(C73A) mutant but not in those transfected with Kv2.1(C710A), suggesting a possible structural alteration in the C-terminal mutant that facilitates membrane insertion. These results show that intracellular N-terminal domains critically regulate phosphorylation of the C-terminal of Kv2.1, and vice versa, suggesting possible new avenues for modifying the apoptotic insertion of these channels during neurodegenerative processes.

No MeSH data available.


Related in: MedlinePlus

Src expression increases p38-dependent phosphorylation of Kv2.1 at S800.A, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10% of total DNA, see Methods), and either Src (15%) or p38 (15%). The membranes with immunoprecipitated Kv2.1 protein were co-probed with anti-Kv2.1 mouse monoclonal antibody and rabbit antibody specific against phosphorylation of Kv2.1 at residue S800. The levels of pKv2.1(S800) in Src- and p38-expressing CHO cells are expressed as the ratio of pKv2.1(S800) to total Kv2.1 protein, and normalized to the same ratio obtained from control cells (value of 1). The data represent mean ± SEM from 6–7 independent experiments (**p < 0.01, two-tailed, unpaired t test). B, Protein samples were harvested from Src- or control vector DNA-expressing CHO cells, the levels of total p38 protein (p38) and phosphorylated p38 protein (p-p38) in equal amounts of total cell lysates were detected by western blotting by using mouse antibody specific against p-p38 and rabbit antibody against total p38 protein. Results (mean ± SEM from 5 independent experiments) show that there is no change of p-p38 levels in Src-overexpressing CHO cells when compared with control cells. C, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%), and either Src (15%) or control vector. Three hours later, transfected cells were treated with a specific p38 MAPK kinase inhibitor, SB 239063 (5 μM). Kv2.1 protein was immunoprecipitated and separated. Immunoblot was performed and quantified as described in Fig 1A. Values (mean ± SEM from 3 independent experiments) represent the ratios of the level of pKv2.1(S800) to total Kv2.1 normalized to their respective controls (-Src, no drug and—Src plus drug; **p < 0.01, two-tailed, paired t test). D, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%) and either Src (15%), p38DN (15%) or control vector. Kv2.1 protein in transfected cells was immunoprecipitated, and quantified as described above. Values (mean ± SEM from 4 independent experiments) represent the ratio of the level of pKv2.1(S800) to total Kv2.1 normalized to respective controls, as in C (*p < 0.01, two-tailed, paired t test).
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pone.0129498.g001: Src expression increases p38-dependent phosphorylation of Kv2.1 at S800.A, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10% of total DNA, see Methods), and either Src (15%) or p38 (15%). The membranes with immunoprecipitated Kv2.1 protein were co-probed with anti-Kv2.1 mouse monoclonal antibody and rabbit antibody specific against phosphorylation of Kv2.1 at residue S800. The levels of pKv2.1(S800) in Src- and p38-expressing CHO cells are expressed as the ratio of pKv2.1(S800) to total Kv2.1 protein, and normalized to the same ratio obtained from control cells (value of 1). The data represent mean ± SEM from 6–7 independent experiments (**p < 0.01, two-tailed, unpaired t test). B, Protein samples were harvested from Src- or control vector DNA-expressing CHO cells, the levels of total p38 protein (p38) and phosphorylated p38 protein (p-p38) in equal amounts of total cell lysates were detected by western blotting by using mouse antibody specific against p-p38 and rabbit antibody against total p38 protein. Results (mean ± SEM from 5 independent experiments) show that there is no change of p-p38 levels in Src-overexpressing CHO cells when compared with control cells. C, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%), and either Src (15%) or control vector. Three hours later, transfected cells were treated with a specific p38 MAPK kinase inhibitor, SB 239063 (5 μM). Kv2.1 protein was immunoprecipitated and separated. Immunoblot was performed and quantified as described in Fig 1A. Values (mean ± SEM from 3 independent experiments) represent the ratios of the level of pKv2.1(S800) to total Kv2.1 normalized to their respective controls (-Src, no drug and—Src plus drug; **p < 0.01, two-tailed, paired t test). D, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%) and either Src (15%), p38DN (15%) or control vector. Kv2.1 protein in transfected cells was immunoprecipitated, and quantified as described above. Values (mean ± SEM from 4 independent experiments) represent the ratio of the level of pKv2.1(S800) to total Kv2.1 normalized to respective controls, as in C (*p < 0.01, two-tailed, paired t test).

Mentions: During apoptosis, Kv2.1 is a substrate of p38 and Src kinases; p38 MAPK mediates phosphorylation of Kv2.1 at residue S800 [21], while Src kinase targets residue Y124 [22, 26]. It has yet to be determined, however, whether there is an interdependence or co-regulation of the pro-apoptotic phosphorylation sites. To begin to explore this question, CHO cells were co-transfected with Kv2.1 as well as either Src or p38-expressing plasmids. Kv2.1 immunoprecipitates were probed with a rabbit antibody recognizing Kv2.1 only when it’s phosphorylated at residue S800 [21]. As expected [28], p38 co-expression significantly increased phosphorylation of Kv2.1(S800) (~1.6-fold over the level of basal phosphorylation; Fig 1A). Notably, Src expression also strongly stimulated Kv2.1(S800) phosphorylation (~2.2-fold increase over the level in control). To investigate whether Src overexpression in CHO cells enhanced p38 MAPK activity to induce the increased phosphorylation of Kv2.1(S800), we measured the level of phosphorylated (i.e. active) p38 in Src-expressing cells but noted no enhancement in the overall signal (Fig 1B). This indicates that Src over-expression, in and of itself, does not stimulate p38 activity, thereby suggesting that the increased phosphorylation of residue S800 in Kv2.1 was due to an alternative mechanism.


Regulation of Pro-Apoptotic Phosphorylation of Kv2.1 K+ Channels.

He K, McCord MC, Hartnett KA, Aizenman E - PLoS ONE (2015)

Src expression increases p38-dependent phosphorylation of Kv2.1 at S800.A, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10% of total DNA, see Methods), and either Src (15%) or p38 (15%). The membranes with immunoprecipitated Kv2.1 protein were co-probed with anti-Kv2.1 mouse monoclonal antibody and rabbit antibody specific against phosphorylation of Kv2.1 at residue S800. The levels of pKv2.1(S800) in Src- and p38-expressing CHO cells are expressed as the ratio of pKv2.1(S800) to total Kv2.1 protein, and normalized to the same ratio obtained from control cells (value of 1). The data represent mean ± SEM from 6–7 independent experiments (**p < 0.01, two-tailed, unpaired t test). B, Protein samples were harvested from Src- or control vector DNA-expressing CHO cells, the levels of total p38 protein (p38) and phosphorylated p38 protein (p-p38) in equal amounts of total cell lysates were detected by western blotting by using mouse antibody specific against p-p38 and rabbit antibody against total p38 protein. Results (mean ± SEM from 5 independent experiments) show that there is no change of p-p38 levels in Src-overexpressing CHO cells when compared with control cells. C, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%), and either Src (15%) or control vector. Three hours later, transfected cells were treated with a specific p38 MAPK kinase inhibitor, SB 239063 (5 μM). Kv2.1 protein was immunoprecipitated and separated. Immunoblot was performed and quantified as described in Fig 1A. Values (mean ± SEM from 3 independent experiments) represent the ratios of the level of pKv2.1(S800) to total Kv2.1 normalized to their respective controls (-Src, no drug and—Src plus drug; **p < 0.01, two-tailed, paired t test). D, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%) and either Src (15%), p38DN (15%) or control vector. Kv2.1 protein in transfected cells was immunoprecipitated, and quantified as described above. Values (mean ± SEM from 4 independent experiments) represent the ratio of the level of pKv2.1(S800) to total Kv2.1 normalized to respective controls, as in C (*p < 0.01, two-tailed, paired t test).
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pone.0129498.g001: Src expression increases p38-dependent phosphorylation of Kv2.1 at S800.A, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10% of total DNA, see Methods), and either Src (15%) or p38 (15%). The membranes with immunoprecipitated Kv2.1 protein were co-probed with anti-Kv2.1 mouse monoclonal antibody and rabbit antibody specific against phosphorylation of Kv2.1 at residue S800. The levels of pKv2.1(S800) in Src- and p38-expressing CHO cells are expressed as the ratio of pKv2.1(S800) to total Kv2.1 protein, and normalized to the same ratio obtained from control cells (value of 1). The data represent mean ± SEM from 6–7 independent experiments (**p < 0.01, two-tailed, unpaired t test). B, Protein samples were harvested from Src- or control vector DNA-expressing CHO cells, the levels of total p38 protein (p38) and phosphorylated p38 protein (p-p38) in equal amounts of total cell lysates were detected by western blotting by using mouse antibody specific against p-p38 and rabbit antibody against total p38 protein. Results (mean ± SEM from 5 independent experiments) show that there is no change of p-p38 levels in Src-overexpressing CHO cells when compared with control cells. C, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%), and either Src (15%) or control vector. Three hours later, transfected cells were treated with a specific p38 MAPK kinase inhibitor, SB 239063 (5 μM). Kv2.1 protein was immunoprecipitated and separated. Immunoblot was performed and quantified as described in Fig 1A. Values (mean ± SEM from 3 independent experiments) represent the ratios of the level of pKv2.1(S800) to total Kv2.1 normalized to their respective controls (-Src, no drug and—Src plus drug; **p < 0.01, two-tailed, paired t test). D, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%) and either Src (15%), p38DN (15%) or control vector. Kv2.1 protein in transfected cells was immunoprecipitated, and quantified as described above. Values (mean ± SEM from 4 independent experiments) represent the ratio of the level of pKv2.1(S800) to total Kv2.1 normalized to respective controls, as in C (*p < 0.01, two-tailed, paired t test).
Mentions: During apoptosis, Kv2.1 is a substrate of p38 and Src kinases; p38 MAPK mediates phosphorylation of Kv2.1 at residue S800 [21], while Src kinase targets residue Y124 [22, 26]. It has yet to be determined, however, whether there is an interdependence or co-regulation of the pro-apoptotic phosphorylation sites. To begin to explore this question, CHO cells were co-transfected with Kv2.1 as well as either Src or p38-expressing plasmids. Kv2.1 immunoprecipitates were probed with a rabbit antibody recognizing Kv2.1 only when it’s phosphorylated at residue S800 [21]. As expected [28], p38 co-expression significantly increased phosphorylation of Kv2.1(S800) (~1.6-fold over the level of basal phosphorylation; Fig 1A). Notably, Src expression also strongly stimulated Kv2.1(S800) phosphorylation (~2.2-fold increase over the level in control). To investigate whether Src overexpression in CHO cells enhanced p38 MAPK activity to induce the increased phosphorylation of Kv2.1(S800), we measured the level of phosphorylated (i.e. active) p38 in Src-expressing cells but noted no enhancement in the overall signal (Fig 1B). This indicates that Src over-expression, in and of itself, does not stimulate p38 activity, thereby suggesting that the increased phosphorylation of residue S800 in Kv2.1 was due to an alternative mechanism.

Bottom Line: Using immunoprecipitated Kv2.1 protein and phospho-specific antibodies, we found that an intact Y124 is required for p38 phosphorylation of S800, and, importantly, that Src phosphorylation of Y124 facilitates the action of the p38 at the S800 residue.Moreover, the actions of Src on Kv2.1 are substantially decreased in the non-phosphorylatable S800A channel mutant.We also observed that mutations of either C73 or C710 residues decreased the p38 phosphorylation at S800 without influencing the actions of Src on tyrosine phosphorylation of Kv2.1.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Pittsburgh School of Medicine, E1456 BST, 3500 Terrace St., Pittsburgh, PA, 15261, United States of America.

ABSTRACT
Caspase activity during apoptosis is inhibited by physiological concentrations of intracellular K+. To enable apoptosis in injured cortical and hippocampal neurons, cellular loss of this cation is facilitated by the insertion of Kv2.1 K+ channels into the plasma membrane via a Zn2+/CaMKII/SNARE-dependent process. Pro-apoptotic membrane insertion of Kv2.1 requires the dual phosphorylation of the channel by Src and p38 at cytoplasmic N- and C-terminal residues Y124 and S800, respectively. In this study, we investigate if these phosphorylation sites are mutually co-regulated, and whether putative N- and C-terminal interactions, possibly enabled by Kv2.1 intracellular cysteine residues C73 and C710, influence the phosphorylation process itself. Studies were performed with recombinant wild type and mutant Kv2.1 expressed in Chinese hamster ovary (CHO) cells. Using immunoprecipitated Kv2.1 protein and phospho-specific antibodies, we found that an intact Y124 is required for p38 phosphorylation of S800, and, importantly, that Src phosphorylation of Y124 facilitates the action of the p38 at the S800 residue. Moreover, the actions of Src on Kv2.1 are substantially decreased in the non-phosphorylatable S800A channel mutant. We also observed that mutations of either C73 or C710 residues decreased the p38 phosphorylation at S800 without influencing the actions of Src on tyrosine phosphorylation of Kv2.1. Surprisingly, however, apoptotic K+ currents were suppressed only in cells expressing the Kv2.1(C73A) mutant but not in those transfected with Kv2.1(C710A), suggesting a possible structural alteration in the C-terminal mutant that facilitates membrane insertion. These results show that intracellular N-terminal domains critically regulate phosphorylation of the C-terminal of Kv2.1, and vice versa, suggesting possible new avenues for modifying the apoptotic insertion of these channels during neurodegenerative processes.

No MeSH data available.


Related in: MedlinePlus