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Regulated binding of importin-? to protein kinase C? in response to apoptotic signals facilitates nuclear import.

ABSTRACT: PKC? translocates into the nucleus in response to apoptotic agents and functions as a potent cell death signal. Cytoplasmic retention of PKC? and its transport into the nucleus are essential for cell homeostasis, but how these processes are regulated is poorly understood. We show that PKC? resides in the cytoplasm in a conformation that precludes binding of importin-?. A structural model of PKC? in the inactive state suggests that the nuclear localization sequence (NLS) is prevented from binding to importin-? through intramolecular contacts between the C2 and catalytic domains. We have previously shown that PKC? is phosphorylated on specific tyrosine residues in response to apoptotic agents. Here, we show that phosphorylation of PKC? at Tyr-64 and Tyr-155 results in a conformational change that allows exposure of the NLS and binding of importin-?. In addition, Hsp90 binds to PKC? with similar kinetics as importin-? and is required for the interaction of importin-? with the NLS. Finally, we elucidate a role for a conserved PPxxP motif, which overlaps the NLS, in nuclear exclusion of PKC?. Mutagenesis of the conserved prolines to alanines enhanced importin-? binding to PKC? and induced its nuclear import in resting cells. Thus, the PPxxP motif is important for maintaining a conformation that facilitates cytosplasmic retention of PKC?. Taken together, this study establishes a novel mechanism that retains PKC? in the cytoplasm of resting cells and regulates its nuclear import in response to apoptotic stimuli.

SUBMITTER: Adwan TS

PROVIDER: S-EPMC3195609 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Regulated binding of importin-α to protein kinase Cδ in response to apoptotic signals facilitates nuclear import.

Adwan Tariq S TS Ohm Angela M AM Jones David N M DNM Humphries Michael J MJ Reyland Mary E ME

The Journal of biological chemistry 20110824 41

PKCδ translocates into the nucleus in response to apoptotic agents and functions as a potent cell death signal. Cytoplasmic retention of PKCδ and its transport into the nucleus are essential for cell homeostasis, but how these processes are regulated is poorly understood. We show that PKCδ resides in the cytoplasm in a conformation that precludes binding of importin-α. A structural model of PKCδ in the inactive state suggests that the nuclear localization sequence (NLS) is prevented from binding ...[more]

PMID: 21865164

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Project description:Human adenoviruses (HAdV) are ubiquitous within the human population and comprise a significant burden of respiratory illnesses worldwide. Pediatric and immunocompromised individuals are at particular risk for developing severe disease; however, no approved antiviral therapies specific to HAdV exist. Ivermectin is an FDA-approved broad-spectrum antiparasitic drug that also exhibits antiviral properties against a diverse range of viruses. Its proposed function is inhibiting the classical protein nuclear import pathway mediated by importin-α (Imp-α) and -β1 (Imp-β1). Many viruses, including HAdV, rely on this host pathway for transport of viral proteins across the nuclear envelope. In this study, we show that ivermectin inhibits HAdV-C5 early gene transcription, early and late protein expression, genome replication, and production of infectious viral progeny. Similarly, ivermectin inhibits genome replication of HAdV-B3, a clinically important pathogen responsible for numerous recent outbreaks. Mechanistically, we show that ivermectin disrupts binding of the viral E1A protein to Imp-α without affecting the interaction between Imp-α and Imp-β1. Our results further extend ivermectin's broad antiviral activity and provide a mechanistic underpinning for its mode of action as an inhibitor of cellular Imp-α/β1-mediated nuclear import.IMPORTANCE Human adenoviruses (HAdVs) represent a ubiquitous and clinically important pathogen without an effective antiviral treatment. HAdV infections typically cause mild symptoms; however, individuals such as children, those with underlying conditions, and those with compromised immune systems can develop severe disseminated disease. Our results demonstrate that ivermectin, an FDA-approved antiparasitic agent, is effective at inhibiting replication of several HAdV types in vitro This is in agreement with the growing body of literature suggesting ivermectin has broad antiviral activity. This study expands our mechanistic knowledge of ivermectin by showing that ivermectin targets the ability of importin-α (Imp-α) to recognize nuclear localization sequences, without effecting the Imp-α/β1 interaction. These data also exemplify the applicability of targeting host factors upon which viruses rely as a viable antiviral strategy.

Dataset Information

Regulated binding of importin-? to protein kinase C? in response to apoptotic signals facilitates nuclear import.

Publications

Regulated binding of importin-α to protein kinase Cδ in response to apoptotic signals facilitates nuclear import.

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