Src expression increases p38-dependent phosphorylation of Kv2.1 at S800.

<p><b>A,</b> CHO cells were co-transfected with plasmid DNAs of Kv2.1 (10% of total DNA, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129498#sec002" target="_blank">Methods</a>), 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 (**<i>p</i> < 0.01, two-tailed, unpaired <i>t</i> test). <b>B,</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. <b>C,</b> 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; **<i>p</i> < 0.01, two-tailed, paired <i>t</i> test). <b>D,</b> 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 <b>C</b> (*<i>p</i> < 0.01, two-tailed, paired <i>t</i> test).</p

Regulation of Pro-Apoptotic Phosphorylation of Kv2.1 K^{+ - Fig 5} Channels

<p>(A) <b>Suppression of apoptotic current enhancement in Kv2.1(C73A)- but not Kv2.1(C710A)-expressing K</b>^<b>+</b><b>channels.</b> Representative whole-cell K⁺ currents and pooled mean ± SEM current densities recorded from Kv2.1-expressing CHO cells without (n = 10) or with (n = 6) 30 μM DTDP, Kv2.1C73A-expressing CHO cells without (n = 9) or with (n = 7) 30 μM DTDP, or Kv2.1C710A-expressing CHO cells without (n = 9) or with (n = 12) 30 μM DTDP. Following DTDP exposure, cells were maintained in the presence of the broad spectrum protease inhibitor 1-3-Boc-aspartyl(Ome)-fluoromethyl ketone (BAF; 10 μM)-containing medium to enhance viability and facilitate electrophysiological recordings [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129498#pone.0129498.ref021" target="_blank">21</a>]. Results show that expression of the C73A channel mutant, but not the C710A mutant, prevents the increase in Kv2.1-mediated K⁺ currents triggered by DTDP. Currents were evoked by a series voltage steps from -80 mV to +80 mV, in 10 mV increments. To determine current density values, steady-state current amplitudes were measured 180 msec after the initiation of the +10 mV step and normalized to cell capacitance. Scale bars: 5 nA, 25 msec; **p<0.01, ***p<0.001, ANOVA/Bonferroni. (B) Kv2.1WT- and mutants-transfected CHO cells were treated with 30 μM DTDP for 10 min and incubated BAF (10 μM)-containing culture medium for 30–60 min. Total lysates were collected and ran for western blots (top). The membranes were co-probed with Kv2.1 and p-Kv2.1(S800) antibodies. GAPDH protein was used as a loading control. The levels of p-Kv2.1(S800) were calculated from the ratio of p-Kv2.1(S800) to total Kv2.1 protein, and then normalized to the levels of p-Kv2.1(S800) in vehicle-treated controls, respectively (bottom). DTDP-induced phosphorylation of both cysteine mutants was inhibited, when compared to wild type Kv2.1 channels. The values represent mean ± SEM from 4 independent experiments (**<i>p</i> < 0.01, ANOVA/Dunnett).</p

Src-induced tyrosine phosphorylation of Kv2.1 in CHO cells is significantly decreased in Kv2.1(S800A) mutants.

<p><b>A,</b> 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>B,</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. <b>C,</b> 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 (***<i>p</i> < 0.001, compared with Kv2.1WT; one sample, two-tailed <i>t</i> test; and ^ΔΔ<i>p</i> < 0.01, two-tailed, paired <i>t</i> test).</p

Kv2.1(Y124F) mutation blocks both Src- and p38-induced phosphorylation of Kv2.1 at S800.

<p><b>A,</b> 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>B,</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 (*<i>p</i> < 0.05 and **<i>p</i> < 0.01, one sample, two-tailed paired <i>t</i> test, vs. 100).</p

N- and C-terminal cysteine residues differentially influence Kv2.1 phosphorylation.

<p><b>A,</b> 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 (****<i>p</i> < 0.0001, compared with Kv2.1WT; one sample, two-tailed <i>t</i> test; and ^ΔΔΔ<i>p</i> < 0.001, two-tailed paired <i>t</i> test). <b>B,</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.</p