Limits...
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.

Show MeSH

Related in: MedlinePlus

A, molecular organization of MLK3 and HCV NS5A. Schematic illustration of the domain structure of the two proteins. Amino acid residue numbers indicated. The sequence of the P2 motif and the mutations introduced to generate the PA2 mutant are illustrated. The black box on NS5A is the N-terminal membrane-associating amphipathic helix. B, structure of bicistronic vectors used for transfection in this study. The construction of these vectors is detailed in the “Experimental Procedures.”
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750171&req=5

Figure 1: A, molecular organization of MLK3 and HCV NS5A. Schematic illustration of the domain structure of the two proteins. Amino acid residue numbers indicated. The sequence of the P2 motif and the mutations introduced to generate the PA2 mutant are illustrated. The black box on NS5A is the N-terminal membrane-associating amphipathic helix. B, structure of bicistronic vectors used for transfection in this study. The construction of these vectors is detailed in the “Experimental Procedures.”

Mentions: Ion channels play a major role in the maintenance of ionic homeostasis. Acute enhancement of voltage-gated K+ channel activity can result in the initiation of apoptosis (10). P38 mitogen-activated protein kinases (p38MAPK) are mitogen-activated protein kinases activated in response to stress stimuli, including cytokines and heat shock, that are involved in cell differentiation and apoptosis (11, 12). It was recently demonstrated that in response to oxidative stress, activated p38MAPK phosphorylates the Kv2.1 protein, which mediates an K+ efflux with a concomitant induction of apoptosis (10, 13). We previously identified that HCV NS5A inhibits the function of Kv2.1 by inhibiting p38MAPK signaling, an effect mediated through the P2 motif. Accordingly, when the P2 motif was mutated (termed PA2) (Fig. 1A) in the context of the HCV isolate JFH1, the virus did not block activation of p38MAPK or Kv2.1, and infected cells were sensitive to oxidant-induced apoptosis (14).


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)

A, molecular organization of MLK3 and HCV NS5A. Schematic illustration of the domain structure of the two proteins. Amino acid residue numbers indicated. The sequence of the P2 motif and the mutations introduced to generate the PA2 mutant are illustrated. The black box on NS5A is the N-terminal membrane-associating amphipathic helix. B, structure of bicistronic vectors used for transfection in this study. The construction of these vectors is detailed in the “Experimental Procedures.”
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750171&req=5

Figure 1: A, molecular organization of MLK3 and HCV NS5A. Schematic illustration of the domain structure of the two proteins. Amino acid residue numbers indicated. The sequence of the P2 motif and the mutations introduced to generate the PA2 mutant are illustrated. The black box on NS5A is the N-terminal membrane-associating amphipathic helix. B, structure of bicistronic vectors used for transfection in this study. The construction of these vectors is detailed in the “Experimental Procedures.”
Mentions: Ion channels play a major role in the maintenance of ionic homeostasis. Acute enhancement of voltage-gated K+ channel activity can result in the initiation of apoptosis (10). P38 mitogen-activated protein kinases (p38MAPK) are mitogen-activated protein kinases activated in response to stress stimuli, including cytokines and heat shock, that are involved in cell differentiation and apoptosis (11, 12). It was recently demonstrated that in response to oxidative stress, activated p38MAPK phosphorylates the Kv2.1 protein, which mediates an K+ efflux with a concomitant induction of apoptosis (10, 13). We previously identified that HCV NS5A inhibits the function of Kv2.1 by inhibiting p38MAPK signaling, an effect mediated through the P2 motif. Accordingly, when the P2 motif was mutated (termed PA2) (Fig. 1A) in the context of the HCV isolate JFH1, the virus did not block activation of p38MAPK or Kv2.1, and infected cells were sensitive to oxidant-induced apoptosis (14).

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