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Modulation of interleukin-1 transcriptional response by the interaction between VRK2 and the JIP1 scaffold protein.

ABSTRACT: BACKGROUND: Cellular biological responses to specific stimulation are determined by a balance among signaling pathways. Protein interactions are likely to modulate these pathways. Vaccinia-related kinase-2 (VRK2) is a novel human kinase that can modulate different signaling pathways. PRINCIPAL FINDINGS: We report that in vivo, the activity of JIP1-JNK complexes is downregulated by VRK2 in response to interleukin-1beta. Also the reduction of endogenous VRK2 with shRNA increases the transcriptional response to IL-1beta. The JIP1 scaffold protein assembles three consecutive members of a given MAPK pathway forming signaling complexes and their signal can be modulated by interactions with regulatory proteins that remain to be identified. Knocking-down JIP1 with siRNA resulted in elimination of the AP1 transcriptional response to IL-1beta. VRK2, a member of novel Ser-Thr kinase family, is able to stably interact with JIP1, TAK1 and MKK7, but not JNK, and can be isolated forming oligomeric complexes with different proportions of TAK1, MKK7beta1 and JNK. JIP1 assembles all these proteins in an oligomeric signalosome. VRK2 binding to the JIP1 signalosome prevents the association of JNK and results in a reduction in its phosphorylation and downregulation of AP1-dependent transcription. CONCLUSIONS/SIGNIFICANCE: This work suggests that the intracellular level of VRK2 protein can modulate the flow through a signaling pathway and alter the response from a receptor that can be distributed by more than one pathway, and thus contribute to the cellular specificity of the response by forming alternative signaling complexes. Furthermore, the effect might be more general and affect other signaling routes assembled on the JIP1 scaffold protein for which a model is proposed.

SUBMITTER: Blanco S

PROVIDER: S-EPMC2243017 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Modulation of interleukin-1 transcriptional response by the interaction between VRK2 and the JIP1 scaffold protein.

Blanco Sandra S Sanz-García Marta M Santos Claudio R CR Lazo Pedro A PA

PloS one 20080220 2

<h4>Background</h4>Cellular biological responses to specific stimulation are determined by a balance among signaling pathways. Protein interactions are likely to modulate these pathways. Vaccinia-related kinase-2 (VRK2) is a novel human kinase that can modulate different signaling pathways.<h4>Principal findings</h4>We report that in vivo, the activity of JIP1-JNK complexes is downregulated by VRK2 in response to interleukin-1beta. Also the reduction of endogenous VRK2 with shRNA increases the t ...[more]

PMID: 18286207

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Project description:The up-regulation of JNK activity is associated with a number of disease states. The JNK-JIP1 interaction represents an attractive target for the inhibition of JNK-mediated signaling. In this study, molecular dynamics simulations have been performed on the apo-JNK1 and the JNK1•L-pepJIP1 and JNK1•D-pepJIP1 complexes to investigate the interaction between the JIP1 peptides and JNK1. Dynamic domain studies based on essential dynamics (ED) analysis of apo-JNK1 and the JNK1•L-pepJIP1 complex have been performed to analyze and compare details of conformational changes, hinge axes, and hinge bending regions in both structures. The activation loop, the ?C helix, and the G loop are found to be highly flexible and to exhibit significant changes in dynamics upon L-pepJIP1 binding. The conformation of the activation loop for the apo state is similar to that of inactive apo-ERK2, while the activation loop in JNK1•L-pepJIP1 complex resembles that of the inactive ERK2 bound with pepHePTP. ED analysis shows that, after the binding of l-pepJIP1, the N- and C-terminal domains of JNK1 display both a closure and a twisting motion centered around the activation loop, which functions as a hinge. In contrast, no domain motion is detected for the apo state for which an open conformation is favored. The present study suggests that L-pepJIP1 regulates the interdomain motions of JNK1 and potentially the active site via an allosteric mechanism. The binding free energies of L-pepJIP1 and D-pepJIP1 to JNK1 are estimated using the molecular mechanics Poisson-Boltzmann and generalized-Born surface area (MM-PB/GBSA) methods. The contribution of each residue at the interaction interface to the binding affinity of L-pepJIP1 with JNK1 has been analyzed by means of computational alanine-scanning mutagenesis and free energy decomposition. Several critical interactions for binding (e.g., Arg156/L-pepJIP1 and Glu329/JNK1) have been identified. The binding free energy calculation indicates that the electrostatic interaction contributes critically to specificity, rather than to binding affinity between the peptide and JNK1. Notably, the binding free energy calculations predict that D-pepJIP1 binding to JNK1 is significantly weaker than the L form, contradicting the previous suggestion that D-pepJIP1 acts as an inhibitor toward JNK1. We have performed experiments using purified JNK1 to confirm that, indeed, D-pepJIP1 does not inhibit the ability of JNK1 to phosphorylate c-Jun in vitro.

Project description:Serine-threonine kinase Akt (also known as PKB, protein kinase B), a core intracellular mediator of cell survival, is involved in various human cancers and has been suggested to play an important role in the regulation of autophagy in mammalian cells. Nonetheless, the physiological function of Akt in the lysosomes is currently unknown. We have reported previously that PtdIns(3)P-dependent lysosomal accumulation of the Akt-Phafin2 complex is a critical step for autophagy induction. Here, to characterize the molecular function of activated Akt in the lysosomes in the process of autophagy, we searched for the molecules that interact with the Akt complex at the lysosomes after induction of autophagy. By time-of-flight-mass spectrometry (TOF/MS) analysis, kinases of the VRK family, a unique serine-threonine family of kinases in the human kinome, were identified. VRK2 interacts with Akt1 and Akt2, but not with Akt3; the C terminus of Akt and the N terminus of VRK2 facilitate the interaction of Akt and VRK2 in mammalian cells. The kinase-dead form of VRK2A (KD VRK2A) failed to interact with Akt in coimmunoprecipitation assays. Bimolecular fluorescence complementation (BiFC) experiments showed that, in the lysosomes, Akt interacted with VRK2A but not with VRK2B or KD VRK2A. Immunofluorescent assays revealed that VRK2 and phosphorylated Akt accumulated in the lysosomes after autophagy induction. WT VRK2A, but not KD VRK2A or VRK2B, facilitated accumulation of phosphorylated Akt in the lysosomes. Downregulation of VRK2 abrogated the lysosomal accumulation of phosphorylated Akt and impaired nuclear localization of TFEB; these events coincided to inhibition of autophagy induction. The VRK2-Akt complex is required for control of lysosomal size, acidification, bacterial degradation, and for viral replication. Moreover, lysosomal VRK2-Akt controls cellular proliferation and mitochondrial outer-membrane stabilization. Given the roles of autophagy in the pathogenesis of human cancer, the current study provides a novel insight into the oncogenic activity of VRK2-Akt complexes in the lysosomes via modulation of autophagy.

Dataset Information

Modulation of interleukin-1 transcriptional response by the interaction between VRK2 and the JIP1 scaffold protein.

Publications

Modulation of interleukin-1 transcriptional response by the interaction between VRK2 and the JIP1 scaffold protein.

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