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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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MLK3 acts as an upstream regulator of Kv2. 1 channel via Ser800 phosphorylation.A, HEK293 cells expressing Kv2.1 were transfected with bicistronic vectors expressing MLK3 (wild type or K144E) together with GFP or NS5A-GFP as shown in Fig. 1B. 16 h post-transfection, Kv2.1 expression was induced by tetracycline treatment, and 8 h later GFP-positive cells were identified by fluorescent microscopy and subjected to electrophysiological recordings. Overexpression of MLK3 wild type augments Kv2.1 channel activity (■), whereas, the K144E kinase-inactive mutant suppressed channel activity (▴). Co-expression of NS5A-GFP abrogated the MLK3 mediated enhancement of Kv2.1 activity (▾). B, comparison of current density measurements at +40 mV. *, p < 0.02, **, p < 0.001, and ***, p < 0.005, unpaired t test. C, representative traces of outward K+ currents in patch-clamp recordings. D, lysates from HEK293 cells expressing Kv2.1 wild type were analyzed by Western blotting with the indicated antibodies. Cells were transfected as follows: lane 1: empty vector, lane 2: NS5A-IRES-GFP, lane 3: MLK3 wild type-IRES-GFP, and lane 4: MLK3 K144E-IRES-GFP.
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Figure 7: MLK3 acts as an upstream regulator of Kv2. 1 channel via Ser800 phosphorylation.A, HEK293 cells expressing Kv2.1 were transfected with bicistronic vectors expressing MLK3 (wild type or K144E) together with GFP or NS5A-GFP as shown in Fig. 1B. 16 h post-transfection, Kv2.1 expression was induced by tetracycline treatment, and 8 h later GFP-positive cells were identified by fluorescent microscopy and subjected to electrophysiological recordings. Overexpression of MLK3 wild type augments Kv2.1 channel activity (■), whereas, the K144E kinase-inactive mutant suppressed channel activity (▴). Co-expression of NS5A-GFP abrogated the MLK3 mediated enhancement of Kv2.1 activity (▾). B, comparison of current density measurements at +40 mV. *, p < 0.02, **, p < 0.001, and ***, p < 0.005, unpaired t test. C, representative traces of outward K+ currents in patch-clamp recordings. D, lysates from HEK293 cells expressing Kv2.1 wild type were analyzed by Western blotting with the indicated antibodies. Cells were transfected as follows: lane 1: empty vector, lane 2: NS5A-IRES-GFP, lane 3: MLK3 wild type-IRES-GFP, and lane 4: MLK3 K144E-IRES-GFP.

Mentions: To investigate whether MLK3 is directly involved in p38MAPK mediated Kv2.1 activation, we transfected tetracycline inducible HEK293 Kv2.1 cells with a bicistronic vectors expressing either wild type or the K144E kinase-inactive mutant of MLK3, together with GFP, and performed whole-cell patch clamp recordings from GFP-positive cells. Exogenous expression of wild type MLK3 significantly enhanced Kv2.1 channel activity (Fig. 7, A–C), while the K144E kinase-inactive mutant showed a dominant negative effect in terms of phosphorylation activation of Kv2.1. To further demonstrate the activation of Kv2.1 in response to MLK3 overexpression, we investigated both p38MAPK activation and Kv2.1 phosphorylation in cells expressing wild type and K144E MLK3. Western blotting analysis demonstrated that MLK3 overexpression led to the activation of both p38MAPK and subsequent Kv2.1 S800 phosphorylation (Fig. 7D, lane 3), which was not observed following transfection of K144E MLK3 (Fig. 7D, lane 4). As NS5A interacts with MLK3 (as shown in Fig. 1), we hypothesized that the inhibition of Kv2.1 activity by NS5A may be mediated through MLK3 inhibition. Accordingly, when cells were transfected with a bicistronic vector expressing wild type MLK3 and an NS5A-GFP fusion protein, the levels of Kv2.1 activity were significantly reduced, suggesting that NS5A inhibits the stimulatory effects of MLK3 on Kv2.1 activity (Fig. 7, A and B).


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)

MLK3 acts as an upstream regulator of Kv2. 1 channel via Ser800 phosphorylation.A, HEK293 cells expressing Kv2.1 were transfected with bicistronic vectors expressing MLK3 (wild type or K144E) together with GFP or NS5A-GFP as shown in Fig. 1B. 16 h post-transfection, Kv2.1 expression was induced by tetracycline treatment, and 8 h later GFP-positive cells were identified by fluorescent microscopy and subjected to electrophysiological recordings. Overexpression of MLK3 wild type augments Kv2.1 channel activity (■), whereas, the K144E kinase-inactive mutant suppressed channel activity (▴). Co-expression of NS5A-GFP abrogated the MLK3 mediated enhancement of Kv2.1 activity (▾). B, comparison of current density measurements at +40 mV. *, p < 0.02, **, p < 0.001, and ***, p < 0.005, unpaired t test. C, representative traces of outward K+ currents in patch-clamp recordings. D, lysates from HEK293 cells expressing Kv2.1 wild type were analyzed by Western blotting with the indicated antibodies. Cells were transfected as follows: lane 1: empty vector, lane 2: NS5A-IRES-GFP, lane 3: MLK3 wild type-IRES-GFP, and lane 4: MLK3 K144E-IRES-GFP.
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Figure 7: MLK3 acts as an upstream regulator of Kv2. 1 channel via Ser800 phosphorylation.A, HEK293 cells expressing Kv2.1 were transfected with bicistronic vectors expressing MLK3 (wild type or K144E) together with GFP or NS5A-GFP as shown in Fig. 1B. 16 h post-transfection, Kv2.1 expression was induced by tetracycline treatment, and 8 h later GFP-positive cells were identified by fluorescent microscopy and subjected to electrophysiological recordings. Overexpression of MLK3 wild type augments Kv2.1 channel activity (■), whereas, the K144E kinase-inactive mutant suppressed channel activity (▴). Co-expression of NS5A-GFP abrogated the MLK3 mediated enhancement of Kv2.1 activity (▾). B, comparison of current density measurements at +40 mV. *, p < 0.02, **, p < 0.001, and ***, p < 0.005, unpaired t test. C, representative traces of outward K+ currents in patch-clamp recordings. D, lysates from HEK293 cells expressing Kv2.1 wild type were analyzed by Western blotting with the indicated antibodies. Cells were transfected as follows: lane 1: empty vector, lane 2: NS5A-IRES-GFP, lane 3: MLK3 wild type-IRES-GFP, and lane 4: MLK3 K144E-IRES-GFP.
Mentions: To investigate whether MLK3 is directly involved in p38MAPK mediated Kv2.1 activation, we transfected tetracycline inducible HEK293 Kv2.1 cells with a bicistronic vectors expressing either wild type or the K144E kinase-inactive mutant of MLK3, together with GFP, and performed whole-cell patch clamp recordings from GFP-positive cells. Exogenous expression of wild type MLK3 significantly enhanced Kv2.1 channel activity (Fig. 7, A–C), while the K144E kinase-inactive mutant showed a dominant negative effect in terms of phosphorylation activation of Kv2.1. To further demonstrate the activation of Kv2.1 in response to MLK3 overexpression, we investigated both p38MAPK activation and Kv2.1 phosphorylation in cells expressing wild type and K144E MLK3. Western blotting analysis demonstrated that MLK3 overexpression led to the activation of both p38MAPK and subsequent Kv2.1 S800 phosphorylation (Fig. 7D, lane 3), which was not observed following transfection of K144E MLK3 (Fig. 7D, lane 4). As NS5A interacts with MLK3 (as shown in Fig. 1), we hypothesized that the inhibition of Kv2.1 activity by NS5A may be mediated through MLK3 inhibition. Accordingly, when cells were transfected with a bicistronic vector expressing wild type MLK3 and an NS5A-GFP fusion protein, the levels of Kv2.1 activity were significantly reduced, suggesting that NS5A inhibits the stimulatory effects of MLK3 on Kv2.1 activity (Fig. 7, A and B).

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