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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

Kv2.1(Y124F) mutation blocks both Src- and p38-induced phosphorylation of Kv2.1 at S800.A, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%) or Kv2.1(Y124F, 30%), and Src (15%) or p38 (15%). The membranes carrying with immunoprecipitated Kv2.1 protein complexes were co-probed with anti-Kv2.1 mouse monoclonal antibody and rabbit antibody specific against serine phosphorylation of Kv2.1 at S800, p-Kv2.1(S800). B, The signal densities of p-Kv2.1(S800) and total Kv2.1 proteins from Y124F mutants in either p38- or Src-expressing CHO cells (panels of Fig 2A) were quantified and the level of p-Kv2.1(S800) was expressed as a ratio of p-Kv2.1(S800) to total Kv2.1 protein and normalized to respective wild type controls (as 100%). The data represents mean ± SEM from 5 independent experiments for each condition (*p < 0.05 and **p < 0.01, one sample, two-tailed paired t test, vs. 100).
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pone.0129498.g002: Kv2.1(Y124F) mutation blocks both Src- and p38-induced phosphorylation of Kv2.1 at S800.A, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%) or Kv2.1(Y124F, 30%), and Src (15%) or p38 (15%). The membranes carrying with immunoprecipitated Kv2.1 protein complexes were co-probed with anti-Kv2.1 mouse monoclonal antibody and rabbit antibody specific against serine phosphorylation of Kv2.1 at S800, p-Kv2.1(S800). B, The signal densities of p-Kv2.1(S800) and total Kv2.1 proteins from Y124F mutants in either p38- or Src-expressing CHO cells (panels of Fig 2A) were quantified and the level of p-Kv2.1(S800) was expressed as a ratio of p-Kv2.1(S800) to total Kv2.1 protein and normalized to respective wild type controls (as 100%). The data represents mean ± SEM from 5 independent experiments for each condition (*p < 0.05 and **p < 0.01, one sample, two-tailed paired t test, vs. 100).

Mentions: To confirm that p38 MAPK was indeed responsible for Src-stimulated phosphorylation of S800, we pre-treated cells with SB 239063 (5 μM), a selective p38 MAPK inhibitor [35]. We observed that SB 239063 almost completely blocked S800 phosphorylation of Kv2.1 induced by Src overexpression (Fig 1C). To further confirm this finding, we transfected a dominant negative p38 construct (p38DN) or its control vector into cells, together with Kv2.1(WT) and Src plasmids. This procedure also markedly reduced Kv2.1(S800) phosphorylation (Fig 1D). As there is some basal, endogenous p38 activity in the CHO cells (Fig 1B), and as also suggested by the presence of phospho-S800 signal under control conditions (see Fig 1A), these results indicate that p38-mediated phosphorylation of Kv2.1 at S800 is likely facilitated by the actions of Src, possibly via the phosphorylation of residue Y124 by this kinase. To evaluate this possibility, we utilized the mutant Kv2.1(Y124F), which lacks the main phosphorylation site for Src [26]. Using this mutant (Fig 2A), we observed a significantly decreased phosphorylation of the S800 residue both in p38- and Src-overexpressing CHO cells (Fig 2B). These results suggest that Src phosphorylation of Y124 positively regulates the phosphorylation of S800 on Kv2.1 by p38. It is noteworthy that in order to generate approximately similar levels of channel expression in these experiments (see S1 Fig), we employed 3 times as much Kv2.1(Y124)-expressing plasmid, when compared to Kv2.1 WT. A similar strategy was employed in all subsequent experiments utilizing Kv2.1 mutant plasmids.


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

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

Kv2.1(Y124F) mutation blocks both Src- and p38-induced phosphorylation of Kv2.1 at S800.A, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%) or Kv2.1(Y124F, 30%), and Src (15%) or p38 (15%). The membranes carrying with immunoprecipitated Kv2.1 protein complexes were co-probed with anti-Kv2.1 mouse monoclonal antibody and rabbit antibody specific against serine phosphorylation of Kv2.1 at S800, p-Kv2.1(S800). B, The signal densities of p-Kv2.1(S800) and total Kv2.1 proteins from Y124F mutants in either p38- or Src-expressing CHO cells (panels of Fig 2A) were quantified and the level of p-Kv2.1(S800) was expressed as a ratio of p-Kv2.1(S800) to total Kv2.1 protein and normalized to respective wild type controls (as 100%). The data represents mean ± SEM from 5 independent experiments for each condition (*p < 0.05 and **p < 0.01, one sample, two-tailed paired t test, vs. 100).
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pone.0129498.g002: Kv2.1(Y124F) mutation blocks both Src- and p38-induced phosphorylation of Kv2.1 at S800.A, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%) or Kv2.1(Y124F, 30%), and Src (15%) or p38 (15%). The membranes carrying with immunoprecipitated Kv2.1 protein complexes were co-probed with anti-Kv2.1 mouse monoclonal antibody and rabbit antibody specific against serine phosphorylation of Kv2.1 at S800, p-Kv2.1(S800). B, The signal densities of p-Kv2.1(S800) and total Kv2.1 proteins from Y124F mutants in either p38- or Src-expressing CHO cells (panels of Fig 2A) were quantified and the level of p-Kv2.1(S800) was expressed as a ratio of p-Kv2.1(S800) to total Kv2.1 protein and normalized to respective wild type controls (as 100%). The data represents mean ± SEM from 5 independent experiments for each condition (*p < 0.05 and **p < 0.01, one sample, two-tailed paired t test, vs. 100).
Mentions: To confirm that p38 MAPK was indeed responsible for Src-stimulated phosphorylation of S800, we pre-treated cells with SB 239063 (5 μM), a selective p38 MAPK inhibitor [35]. We observed that SB 239063 almost completely blocked S800 phosphorylation of Kv2.1 induced by Src overexpression (Fig 1C). To further confirm this finding, we transfected a dominant negative p38 construct (p38DN) or its control vector into cells, together with Kv2.1(WT) and Src plasmids. This procedure also markedly reduced Kv2.1(S800) phosphorylation (Fig 1D). As there is some basal, endogenous p38 activity in the CHO cells (Fig 1B), and as also suggested by the presence of phospho-S800 signal under control conditions (see Fig 1A), these results indicate that p38-mediated phosphorylation of Kv2.1 at S800 is likely facilitated by the actions of Src, possibly via the phosphorylation of residue Y124 by this kinase. To evaluate this possibility, we utilized the mutant Kv2.1(Y124F), which lacks the main phosphorylation site for Src [26]. Using this mutant (Fig 2A), we observed a significantly decreased phosphorylation of the S800 residue both in p38- and Src-overexpressing CHO cells (Fig 2B). These results suggest that Src phosphorylation of Y124 positively regulates the phosphorylation of S800 on Kv2.1 by p38. It is noteworthy that in order to generate approximately similar levels of channel expression in these experiments (see S1 Fig), we employed 3 times as much Kv2.1(Y124)-expressing plasmid, when compared to Kv2.1 WT. A similar strategy was employed in all subsequent experiments utilizing Kv2.1 mutant plasmids.

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