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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-induced tyrosine phosphorylation of Kv2.1 in CHO cells is significantly decreased in Kv2.1(S800A) mutants.A, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%), Kv2.1(S800A) (10%), Src (15%) and vector controls. Protein samples were collected 24 h later and Kv2.1 protein was immunoprecipitated and transferred onto nitrocellulose membranes. The membranes were co-probed with mouse anti-phospho-tyrosine monoclonal antibody, p-Kv2.1(Tyr) and rabbit polyclonal antibody specific against total Kv2.1 (Kv2.1). B, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%), Kv2.1(S800E) (10%), Src (15%) and vector controls, followed by experimental procedures described in Fig 3A. C, The signal densities of p-Kv2.1(Tyr) and total Kv2.1 proteins from Kv2.1WT, Kv2.1(S800A) and Kv2.1(S800E) were quantified as described earlier. The level of p-Kv2.1(Tyr) was expressed as a ratio of p-Kv2.1(Tyr) to total Kv2.1 protein and normalized to the tyrosine phosphorylation level of Kv2.1WT in CHO cells without Src overexpression. The data represents mean ± SEM from 4 independent experiments (***p < 0.001, compared with Kv2.1WT; one sample, two-tailed t test; and ΔΔp < 0.01, two-tailed, paired t test).
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pone.0129498.g003: Src-induced tyrosine phosphorylation of Kv2.1 in CHO cells is significantly decreased in Kv2.1(S800A) mutants.A, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%), Kv2.1(S800A) (10%), Src (15%) and vector controls. Protein samples were collected 24 h later and Kv2.1 protein was immunoprecipitated and transferred onto nitrocellulose membranes. The membranes were co-probed with mouse anti-phospho-tyrosine monoclonal antibody, p-Kv2.1(Tyr) and rabbit polyclonal antibody specific against total Kv2.1 (Kv2.1). B, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%), Kv2.1(S800E) (10%), Src (15%) and vector controls, followed by experimental procedures described in Fig 3A. C, The signal densities of p-Kv2.1(Tyr) and total Kv2.1 proteins from Kv2.1WT, Kv2.1(S800A) and Kv2.1(S800E) were quantified as described earlier. The level of p-Kv2.1(Tyr) was expressed as a ratio of p-Kv2.1(Tyr) to total Kv2.1 protein and normalized to the tyrosine phosphorylation level of Kv2.1WT in CHO cells without Src overexpression. The data represents mean ± SEM from 4 independent experiments (***p < 0.001, compared with Kv2.1WT; one sample, two-tailed t test; and ΔΔp < 0.01, two-tailed, paired t test).

Mentions: Next, we investigated the reverse situation, namely, whether changes in the S800 site would affect Src-mediated phosphorylation of Kv2.1. Although we have not been able to generate successfully a phospho-specific antibody to residue Y124 [21], it has been previously demonstrated that a majority (>70%) of the phospho-tyrosine signal of immunoprecipitated Kv2.1 is derived from that residue [26]. These experiments were simplified by the fact that mutant Kv2.1(S800A) channels are insensitive to p38 phosphorylation, while Kv2.1(S800E) effectively mimics the p38-phosphorylated state [22]. In contrast to the previously noted basal p38 phosphorylation of residue S800 (Fig 1A), CHO cells appear not to show resting phospho-tyrosine activity, at least with regards to immunoprecipitated Kv2.1 protein, nor does co-expression of p38 increase phospho-tyrosine signal (not shown). However, co-expression of Src induced a remarkable increase in tyrosine phosphorylation of wild-type Kv2.1 (Fig 3A and 3B). The levels of Src-induced phospho-tyrosine activity were significantly reduced in Kv2.1(S800A) mutant channels (Fig 3A), while phospho-tyrosine levels in Kv2.1(S800E) channels were comparable to wild type (Fig 3B). These data suggest that Src-related tyrosine phosphorylation of Kv2.1 can be modulated by p38-mediated phosphorylation at S800, again pointing to mutual regulatory activities at the pro-apoptotic phosphorylation sites on Kv2.1.


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

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

Src-induced tyrosine phosphorylation of Kv2.1 in CHO cells is significantly decreased in Kv2.1(S800A) mutants.A, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%), Kv2.1(S800A) (10%), Src (15%) and vector controls. Protein samples were collected 24 h later and Kv2.1 protein was immunoprecipitated and transferred onto nitrocellulose membranes. The membranes were co-probed with mouse anti-phospho-tyrosine monoclonal antibody, p-Kv2.1(Tyr) and rabbit polyclonal antibody specific against total Kv2.1 (Kv2.1). B, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%), Kv2.1(S800E) (10%), Src (15%) and vector controls, followed by experimental procedures described in Fig 3A. C, The signal densities of p-Kv2.1(Tyr) and total Kv2.1 proteins from Kv2.1WT, Kv2.1(S800A) and Kv2.1(S800E) were quantified as described earlier. The level of p-Kv2.1(Tyr) was expressed as a ratio of p-Kv2.1(Tyr) to total Kv2.1 protein and normalized to the tyrosine phosphorylation level of Kv2.1WT in CHO cells without Src overexpression. The data represents mean ± SEM from 4 independent experiments (***p < 0.001, compared with Kv2.1WT; one sample, two-tailed t test; and ΔΔp < 0.01, two-tailed, paired t test).
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pone.0129498.g003: Src-induced tyrosine phosphorylation of Kv2.1 in CHO cells is significantly decreased in Kv2.1(S800A) mutants.A, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%), Kv2.1(S800A) (10%), Src (15%) and vector controls. Protein samples were collected 24 h later and Kv2.1 protein was immunoprecipitated and transferred onto nitrocellulose membranes. The membranes were co-probed with mouse anti-phospho-tyrosine monoclonal antibody, p-Kv2.1(Tyr) and rabbit polyclonal antibody specific against total Kv2.1 (Kv2.1). B, CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10%), Kv2.1(S800E) (10%), Src (15%) and vector controls, followed by experimental procedures described in Fig 3A. C, The signal densities of p-Kv2.1(Tyr) and total Kv2.1 proteins from Kv2.1WT, Kv2.1(S800A) and Kv2.1(S800E) were quantified as described earlier. The level of p-Kv2.1(Tyr) was expressed as a ratio of p-Kv2.1(Tyr) to total Kv2.1 protein and normalized to the tyrosine phosphorylation level of Kv2.1WT in CHO cells without Src overexpression. The data represents mean ± SEM from 4 independent experiments (***p < 0.001, compared with Kv2.1WT; one sample, two-tailed t test; and ΔΔp < 0.01, two-tailed, paired t test).
Mentions: Next, we investigated the reverse situation, namely, whether changes in the S800 site would affect Src-mediated phosphorylation of Kv2.1. Although we have not been able to generate successfully a phospho-specific antibody to residue Y124 [21], it has been previously demonstrated that a majority (>70%) of the phospho-tyrosine signal of immunoprecipitated Kv2.1 is derived from that residue [26]. These experiments were simplified by the fact that mutant Kv2.1(S800A) channels are insensitive to p38 phosphorylation, while Kv2.1(S800E) effectively mimics the p38-phosphorylated state [22]. In contrast to the previously noted basal p38 phosphorylation of residue S800 (Fig 1A), CHO cells appear not to show resting phospho-tyrosine activity, at least with regards to immunoprecipitated Kv2.1 protein, nor does co-expression of p38 increase phospho-tyrosine signal (not shown). However, co-expression of Src induced a remarkable increase in tyrosine phosphorylation of wild-type Kv2.1 (Fig 3A and 3B). The levels of Src-induced phospho-tyrosine activity were significantly reduced in Kv2.1(S800A) mutant channels (Fig 3A), while phospho-tyrosine levels in Kv2.1(S800E) channels were comparable to wild type (Fig 3B). These data suggest that Src-related tyrosine phosphorylation of Kv2.1 can be modulated by p38-mediated phosphorylation at S800, again pointing to mutual regulatory activities at the pro-apoptotic phosphorylation sites on Kv2.1.

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