Limits...
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

N- and C-terminal cysteine residues differentially influence Kv2.1 phosphorylation.A, CHO cells were co-transfected with plasmid DNAs of p38 (15%), and Kv2.1(WT) (10%), Kv2.1(C73A) (30%), or Kv2.1(C710A) (30%). The membranes with separated immunoprecipitated Kv2.1 protein complexes were co-probed with mouse anti-Kv2.1 monoclonal antibody and rabbit polyclonal antibody specific against serine phosphorylation of Kv2.1 at S800, p-Kv2.1(S800). The level of p-Kv2.1(S800) was calculated from the ratio of p-Kv2.1(S800) to total Kv2.1 protein, and then normalized to the level of p-Kv2.1(S800, WT) in p38-transfected CHO cells. The values represent mean ± SEM from 7 independent experiments (****p < 0.0001, compared with Kv2.1WT; one sample, two-tailed t test; and ΔΔΔp < 0.001, two-tailed paired t test). B, CHO cells were co-transfected with plasmid DNAs of Src (15%), and Kv2.1 (WT, 10%), Kv2.1(C73A, 30%), or Kv2.1(C710A, 30%). Immunoblot was co-probed with rabbit anti-Kv2.1 polyclonal antibody (Kv2.1) and mouse anti-phosphotyrosine antibody, p-Kv2.1(Tyr). The signal densities of p-Kv2.1(Tyr) and total Kv2.1 proteins from Kv2.1WT, Kv2.1(C73A) and Kv2.1(C710A) were quantified as described above. The level of p-Kv2.1(Tyr) was calculated as the ratio of pKv2.1(Tyr) to total Kv2.1 protein and normalized to tyrosine phosphorylation of Kv2.1WT in CHO cells with Src overexpression. Similar p-Kv2.1(Tyr) levels were detected in Src-expressing CHO cells in WT, C73A and C710A groups.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482604&req=5

pone.0129498.g004: N- and C-terminal cysteine residues differentially influence Kv2.1 phosphorylation.A, CHO cells were co-transfected with plasmid DNAs of p38 (15%), and Kv2.1(WT) (10%), Kv2.1(C73A) (30%), or Kv2.1(C710A) (30%). The membranes with separated immunoprecipitated Kv2.1 protein complexes were co-probed with mouse anti-Kv2.1 monoclonal antibody and rabbit polyclonal antibody specific against serine phosphorylation of Kv2.1 at S800, p-Kv2.1(S800). The level of p-Kv2.1(S800) was calculated from the ratio of p-Kv2.1(S800) to total Kv2.1 protein, and then normalized to the level of p-Kv2.1(S800, WT) in p38-transfected CHO cells. The values represent mean ± SEM from 7 independent experiments (****p < 0.0001, compared with Kv2.1WT; one sample, two-tailed t test; and ΔΔΔp < 0.001, two-tailed paired t test). B, CHO cells were co-transfected with plasmid DNAs of Src (15%), and Kv2.1 (WT, 10%), Kv2.1(C73A, 30%), or Kv2.1(C710A, 30%). Immunoblot was co-probed with rabbit anti-Kv2.1 polyclonal antibody (Kv2.1) and mouse anti-phosphotyrosine antibody, p-Kv2.1(Tyr). The signal densities of p-Kv2.1(Tyr) and total Kv2.1 proteins from Kv2.1WT, Kv2.1(C73A) and Kv2.1(C710A) were quantified as described above. The level of p-Kv2.1(Tyr) was calculated as the ratio of pKv2.1(Tyr) to total Kv2.1 protein and normalized to tyrosine phosphorylation of Kv2.1WT in CHO cells with Src overexpression. Similar p-Kv2.1(Tyr) levels were detected in Src-expressing CHO cells in WT, C73A and C710A groups.

Mentions: A recent study showed that a cysteine residue (C73) located in the N-terminus of Kv2.1 is involved in the process of neuronal apoptosis associated with oxidation of the channel. Indeed, mutating C73 to an alanine was sufficient to induce cytoprotection [29]. Furthermore, C73 was proposed by Sesti and co-workers to interact with cysteine 710 (C710) of the Kv2.1 C-terminus through an inter- or intra-subunit disulfide bond [29]. As such, we tested whether C73 and C710 may influence the phosphorylation of pro-apoptotic residues of Kv2.1. First, we compared the effects of p38-induced S800 phosphorylation in wild type and cysteine mutant channels. We observed that phosphorylation of S800 by p38 co-expression was substantially reduced in CHO cells transfected with either Kv2.1(C73A) or Kv2.1(710A), the effects being markedly more pronounced in the C73A mutant (Fig 4A). In contrast, tyrosine phosphorylation of Kv2.1 induced by Src co-expression in the cysteine mutant channels was relatively comparable to Kv2.1(WT) (Fig 4B). These results suggest that mutations at cysteine residues that promote putative interactions between the N and C termini of Kv2.1 can have pronounced effects on p38, but not Src phosphorylation of the channel.


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

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

N- and C-terminal cysteine residues differentially influence Kv2.1 phosphorylation.A, CHO cells were co-transfected with plasmid DNAs of p38 (15%), and Kv2.1(WT) (10%), Kv2.1(C73A) (30%), or Kv2.1(C710A) (30%). The membranes with separated immunoprecipitated Kv2.1 protein complexes were co-probed with mouse anti-Kv2.1 monoclonal antibody and rabbit polyclonal antibody specific against serine phosphorylation of Kv2.1 at S800, p-Kv2.1(S800). The level of p-Kv2.1(S800) was calculated from the ratio of p-Kv2.1(S800) to total Kv2.1 protein, and then normalized to the level of p-Kv2.1(S800, WT) in p38-transfected CHO cells. The values represent mean ± SEM from 7 independent experiments (****p < 0.0001, compared with Kv2.1WT; one sample, two-tailed t test; and ΔΔΔp < 0.001, two-tailed paired t test). B, CHO cells were co-transfected with plasmid DNAs of Src (15%), and Kv2.1 (WT, 10%), Kv2.1(C73A, 30%), or Kv2.1(C710A, 30%). Immunoblot was co-probed with rabbit anti-Kv2.1 polyclonal antibody (Kv2.1) and mouse anti-phosphotyrosine antibody, p-Kv2.1(Tyr). The signal densities of p-Kv2.1(Tyr) and total Kv2.1 proteins from Kv2.1WT, Kv2.1(C73A) and Kv2.1(C710A) were quantified as described above. The level of p-Kv2.1(Tyr) was calculated as the ratio of pKv2.1(Tyr) to total Kv2.1 protein and normalized to tyrosine phosphorylation of Kv2.1WT in CHO cells with Src overexpression. Similar p-Kv2.1(Tyr) levels were detected in Src-expressing CHO cells in WT, C73A and C710A groups.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482604&req=5

pone.0129498.g004: N- and C-terminal cysteine residues differentially influence Kv2.1 phosphorylation.A, CHO cells were co-transfected with plasmid DNAs of p38 (15%), and Kv2.1(WT) (10%), Kv2.1(C73A) (30%), or Kv2.1(C710A) (30%). The membranes with separated immunoprecipitated Kv2.1 protein complexes were co-probed with mouse anti-Kv2.1 monoclonal antibody and rabbit polyclonal antibody specific against serine phosphorylation of Kv2.1 at S800, p-Kv2.1(S800). The level of p-Kv2.1(S800) was calculated from the ratio of p-Kv2.1(S800) to total Kv2.1 protein, and then normalized to the level of p-Kv2.1(S800, WT) in p38-transfected CHO cells. The values represent mean ± SEM from 7 independent experiments (****p < 0.0001, compared with Kv2.1WT; one sample, two-tailed t test; and ΔΔΔp < 0.001, two-tailed paired t test). B, CHO cells were co-transfected with plasmid DNAs of Src (15%), and Kv2.1 (WT, 10%), Kv2.1(C73A, 30%), or Kv2.1(C710A, 30%). Immunoblot was co-probed with rabbit anti-Kv2.1 polyclonal antibody (Kv2.1) and mouse anti-phosphotyrosine antibody, p-Kv2.1(Tyr). The signal densities of p-Kv2.1(Tyr) and total Kv2.1 proteins from Kv2.1WT, Kv2.1(C73A) and Kv2.1(C710A) were quantified as described above. The level of p-Kv2.1(Tyr) was calculated as the ratio of pKv2.1(Tyr) to total Kv2.1 protein and normalized to tyrosine phosphorylation of Kv2.1WT in CHO cells with Src overexpression. Similar p-Kv2.1(Tyr) levels were detected in Src-expressing CHO cells in WT, C73A and C710A groups.
Mentions: A recent study showed that a cysteine residue (C73) located in the N-terminus of Kv2.1 is involved in the process of neuronal apoptosis associated with oxidation of the channel. Indeed, mutating C73 to an alanine was sufficient to induce cytoprotection [29]. Furthermore, C73 was proposed by Sesti and co-workers to interact with cysteine 710 (C710) of the Kv2.1 C-terminus through an inter- or intra-subunit disulfide bond [29]. As such, we tested whether C73 and C710 may influence the phosphorylation of pro-apoptotic residues of Kv2.1. First, we compared the effects of p38-induced S800 phosphorylation in wild type and cysteine mutant channels. We observed that phosphorylation of S800 by p38 co-expression was substantially reduced in CHO cells transfected with either Kv2.1(C73A) or Kv2.1(710A), the effects being markedly more pronounced in the C73A mutant (Fig 4A). In contrast, tyrosine phosphorylation of Kv2.1 induced by Src co-expression in the cysteine mutant channels was relatively comparable to Kv2.1(WT) (Fig 4B). These results suggest that mutations at cysteine residues that promote putative interactions between the N and C termini of Kv2.1 can have pronounced effects on p38, but not Src phosphorylation of the channel.

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