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Hepatitis C virus NS5A inhibits mixed lineage kinase 3 to block apoptosis.

Amako Y, Igloi Z, Mankouri J, Kazlauskas A, Saksela K, Dallas M, Peers C, Harris M - J. Biol. Chem. (2013)

Bottom Line: We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1.An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis.We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K(+) homeostasis.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.

ABSTRACT
Hepatitis C virus (HCV) infection results in the activation of numerous stress responses including oxidative stress, with the potential to induce an apoptotic state. Previously we have shown that HCV attenuates the stress-induced, p38MAPK-mediated up-regulation of the K(+) channel Kv2.1, to maintain the survival of infected cells in the face of cellular stress. We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1. In this study, we investigated the host cell proteins targeted by NS5A to mediate Kv2.1 inhibition. We screened a phage-display library expressing the entire complement of human SH3 domains for novel NS5A-host cell interactions. This analysis identified mixed lineage kinase 3 (MLK3) as a putative NS5A interacting partner. MLK3 is a serine/threonine protein kinase that is a member of the MAPK kinase kinase (MAP3K) family and activates p38MAPK. An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis. We further demonstrate a novel role of MLK3 in the modulation of Kv2.1 activity, whereby MLK3 overexpression leads to the up-regulation of channel activity. Accordingly, coexpression of NS5A suppressed this stimulation. Additionally we demonstrate that overexpression of MLK3 induced apoptosis, which was also counteracted by NS5A. We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K(+) homeostasis.

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Kv2.1 ion channel activity is regulated by serine 800 phosphorylation. Whole-cell patch clamp recordings of Kv2.1 K+ currents. HEK293 cell lines expressing wild type Kv2.1 (A), S800A (non-phosphorylatable mutant: B) or S800E (phosphomimetic mutant: C) were subjected to whole cell patch clamp in the presence (○) or absence (●) of the oxidant DTDP (n = 3). Insets show representative traces of K+ currents in patch-clamp recordings obtained by step depolarizations applied from a holding potential of −70 mV to between −100 mV and +60 mV, in 10-mV increments. Top; trace without DTDP stimulation, below: with DTDP stimulation (D) HEK293 Kv2.1 cells were incubated with tetracycline to induce Kv2.1 expression, and/or DTDP to induce oxidative stress. Proteins expressed on the plasma membrane of HEK293 Kv2.1 cells were biotin-labeled and harvested samples were probed by Western blot with anti-Kv2.1 antibody (top panel). Whole cell lysates were also probed with the total Kv2.1 (middle). Comparable amount of Kv2.1 proteins were expressed following addition of tetracycline. GAPDH detection was performed as a control (bottom).
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Figure 4: Kv2.1 ion channel activity is regulated by serine 800 phosphorylation. Whole-cell patch clamp recordings of Kv2.1 K+ currents. HEK293 cell lines expressing wild type Kv2.1 (A), S800A (non-phosphorylatable mutant: B) or S800E (phosphomimetic mutant: C) were subjected to whole cell patch clamp in the presence (○) or absence (●) of the oxidant DTDP (n = 3). Insets show representative traces of K+ currents in patch-clamp recordings obtained by step depolarizations applied from a holding potential of −70 mV to between −100 mV and +60 mV, in 10-mV increments. Top; trace without DTDP stimulation, below: with DTDP stimulation (D) HEK293 Kv2.1 cells were incubated with tetracycline to induce Kv2.1 expression, and/or DTDP to induce oxidative stress. Proteins expressed on the plasma membrane of HEK293 Kv2.1 cells were biotin-labeled and harvested samples were probed by Western blot with anti-Kv2.1 antibody (top panel). Whole cell lysates were also probed with the total Kv2.1 (middle). Comparable amount of Kv2.1 proteins were expressed following addition of tetracycline. GAPDH detection was performed as a control (bottom).

Mentions: As we previously reported, the inhibition of both apoptosis and the activity of the Kv2.1 channel were dependent on the P2 motif within NS5A (14). Taken together with the results shown in Figs. 2 and 3, we therefore considered that MLK3 might also contribute to Kv2.1 regulation. To test this, and further elucidate the mechanism by which NS5A inhibited Kv2.1 activity, we established a novel model cell line for electrophysiological measurement of Kv2.1 activity and its regulation. Stable HEK293 cell lines expressing either wildtype Kv2.1, a non-phosphorylatable S800A, or a phosphomimetic S800E mutant, under the control of tetracycline-regulatable promoter were produced. For cells expressing wild type Kv2.1, we were able to record a typical voltage gated outward K+ current upon tetracycline induction (Fig. 4A) that could be stimulated by oxidant (DTDP) treatment. Currents were augmented 3.5–4 fold by DTDP treatment over the voltage range +10 mV to +60 mV.


Hepatitis C virus NS5A inhibits mixed lineage kinase 3 to block apoptosis.

Amako Y, Igloi Z, Mankouri J, Kazlauskas A, Saksela K, Dallas M, Peers C, Harris M - J. Biol. Chem. (2013)

Kv2.1 ion channel activity is regulated by serine 800 phosphorylation. Whole-cell patch clamp recordings of Kv2.1 K+ currents. HEK293 cell lines expressing wild type Kv2.1 (A), S800A (non-phosphorylatable mutant: B) or S800E (phosphomimetic mutant: C) were subjected to whole cell patch clamp in the presence (○) or absence (●) of the oxidant DTDP (n = 3). Insets show representative traces of K+ currents in patch-clamp recordings obtained by step depolarizations applied from a holding potential of −70 mV to between −100 mV and +60 mV, in 10-mV increments. Top; trace without DTDP stimulation, below: with DTDP stimulation (D) HEK293 Kv2.1 cells were incubated with tetracycline to induce Kv2.1 expression, and/or DTDP to induce oxidative stress. Proteins expressed on the plasma membrane of HEK293 Kv2.1 cells were biotin-labeled and harvested samples were probed by Western blot with anti-Kv2.1 antibody (top panel). Whole cell lysates were also probed with the total Kv2.1 (middle). Comparable amount of Kv2.1 proteins were expressed following addition of tetracycline. GAPDH detection was performed as a control (bottom).
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Related In: Results  -  Collection

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Figure 4: Kv2.1 ion channel activity is regulated by serine 800 phosphorylation. Whole-cell patch clamp recordings of Kv2.1 K+ currents. HEK293 cell lines expressing wild type Kv2.1 (A), S800A (non-phosphorylatable mutant: B) or S800E (phosphomimetic mutant: C) were subjected to whole cell patch clamp in the presence (○) or absence (●) of the oxidant DTDP (n = 3). Insets show representative traces of K+ currents in patch-clamp recordings obtained by step depolarizations applied from a holding potential of −70 mV to between −100 mV and +60 mV, in 10-mV increments. Top; trace without DTDP stimulation, below: with DTDP stimulation (D) HEK293 Kv2.1 cells were incubated with tetracycline to induce Kv2.1 expression, and/or DTDP to induce oxidative stress. Proteins expressed on the plasma membrane of HEK293 Kv2.1 cells were biotin-labeled and harvested samples were probed by Western blot with anti-Kv2.1 antibody (top panel). Whole cell lysates were also probed with the total Kv2.1 (middle). Comparable amount of Kv2.1 proteins were expressed following addition of tetracycline. GAPDH detection was performed as a control (bottom).
Mentions: As we previously reported, the inhibition of both apoptosis and the activity of the Kv2.1 channel were dependent on the P2 motif within NS5A (14). Taken together with the results shown in Figs. 2 and 3, we therefore considered that MLK3 might also contribute to Kv2.1 regulation. To test this, and further elucidate the mechanism by which NS5A inhibited Kv2.1 activity, we established a novel model cell line for electrophysiological measurement of Kv2.1 activity and its regulation. Stable HEK293 cell lines expressing either wildtype Kv2.1, a non-phosphorylatable S800A, or a phosphomimetic S800E mutant, under the control of tetracycline-regulatable promoter were produced. For cells expressing wild type Kv2.1, we were able to record a typical voltage gated outward K+ current upon tetracycline induction (Fig. 4A) that could be stimulated by oxidant (DTDP) treatment. Currents were augmented 3.5–4 fold by DTDP treatment over the voltage range +10 mV to +60 mV.

Bottom Line: We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1.An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis.We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K(+) homeostasis.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.

ABSTRACT
Hepatitis C virus (HCV) infection results in the activation of numerous stress responses including oxidative stress, with the potential to induce an apoptotic state. Previously we have shown that HCV attenuates the stress-induced, p38MAPK-mediated up-regulation of the K(+) channel Kv2.1, to maintain the survival of infected cells in the face of cellular stress. We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1. In this study, we investigated the host cell proteins targeted by NS5A to mediate Kv2.1 inhibition. We screened a phage-display library expressing the entire complement of human SH3 domains for novel NS5A-host cell interactions. This analysis identified mixed lineage kinase 3 (MLK3) as a putative NS5A interacting partner. MLK3 is a serine/threonine protein kinase that is a member of the MAPK kinase kinase (MAP3K) family and activates p38MAPK. An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis. We further demonstrate a novel role of MLK3 in the modulation of Kv2.1 activity, whereby MLK3 overexpression leads to the up-regulation of channel activity. Accordingly, coexpression of NS5A suppressed this stimulation. Additionally we demonstrate that overexpression of MLK3 induced apoptosis, which was also counteracted by NS5A. We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K(+) homeostasis.

Show MeSH
Related in: MedlinePlus