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Host Protein Kinase C⍺: The novel Mitogen Activated Protein Kinase (MAPK) specific scaffold regulating nuclear export

Indrani Das Jana¹, Soumik Dey¹, Manoj Si¹

¹Department of Bioscience and Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, India.

|December 31, 2025

Abstract:

Host protein kinase C (PKC) isoforms are well known modulators of different steps of influenza virus replication cycle. PKC⍺ was reported to activate the Rapidly Accelerated Fibrosarcoma (Raf)/ Mitogen-activated protein kinase kinase (MEK)/ Extracellular signal-regulated kinase (ERK)- mitogen-activated protein kinase (MAPK) pathway to promote nuclear export of influenza virus ribonucleoprotein complexes (RNPs). However, the molecular mechanism by which PKC⍺ activates specific members of the MAPK cascade and thereby facilitate virus replication, has never been investigated. Here we unravel the novel role of PKC⍺ as a MAPK-specific scaffold to bridge stable kinase-substrate interaction between ERK2 with influenza virus nucleoprotein NP, the major constituent of RNP. Using analogue sensitive kinase, we show that ERK2 can directly phosphorylate NP at specific serine-threonine residues, which promote vRNP nuclear export and are indispensable for virus propagation. PKC⍺ not only activates MAPK cascade, but also participates in stable interactions with the upstream kinase MEK1, effector kinase ERK2, and the substrate NP, thereby forming a multiprotein complex that regulate ERK2 activation, substrate recognition and subsequent phosphorylation events. This multiprotein complex localizes in the nucleus early during infection but eventually moves into cytoplasm at later stages of the viral life cycle. Overexpression of a dominant negative variant of PKC⍺ blocks this complex formation, vRNP export and progeny virus production, thereby establishing PKC⍺ as a key regulator of influenza virus replication. In summary, our results advance the molecular level understanding of the cross-talk between PKC⍺ and MAPK pathway supporting influenza A and B virus replication.

Insights

Protein Kinase C alpha (PKCα) acts as a scaffold protein, bridging ERK2 and influenza virus nucleoprotein (NP) to promote viral replication. This interaction is crucial for vRNP nuclear export and virus propagation.

Area of Science:

Virology
Molecular Biology
Cellular Biology

Background:

Host protein kinase C (PKC) isoforms regulate influenza virus replication.
PKCα activates the Raf/MEK/ERK-MAPK pathway, promoting viral ribonucleoprotein (vRNP) nuclear export.
The precise mechanism of PKCα-mediated MAPK activation in influenza virus replication remains unclear.

Purpose of the Study:

To elucidate the molecular mechanism of PKCα in activating the MAPK cascade during influenza virus replication.
To investigate the role of PKCα as a scaffold protein in kinase-substrate interactions.
To determine the significance of PKCα-mediated NP phosphorylation for virus propagation.

Main Methods:

Utilized analogue-sensitive kinase technology to identify direct phosphorylation sites on influenza virus nucleoprotein (NP) by ERK2.
Investigated the formation and localization of a multiprotein complex involving PKCα, MEK1, ERK2, and NP.
Employed dominant-negative variants of PKCα to assess its role in complex formation, vRNP export, and virus production.

Main Results:

PKCα functions as a MAPK-specific scaffold, facilitating stable interaction between ERK2 and influenza virus NP.
ERK2 directly phosphorylates NP at specific residues, promoting vRNP nuclear export and enabling virus propagation.
A PKCα-containing multiprotein complex regulates ERK2 activation and NP phosphorylation, localizing to the nucleus then cytoplasm.
Inhibition of PKCα disrupts complex formation, vRNP export, and progeny virus production.

Conclusions:

PKCα is a critical regulator of influenza virus replication by acting as a scaffold for the ERK2-NP interaction.
This study reveals a novel mechanism for cross-talk between PKCα and the MAPK pathway in supporting influenza A and B virus replication.
Targeting the PKCα-MAPK pathway could offer new strategies for antiviral therapies.

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