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Identification of arrestin-3-specific residues necessary for JNK3 kinase activation.

ABSTRACT: Arrestins bind active phosphorylated G protein-coupled receptors, blocking G protein activation and channeling the signaling to G protein-independent pathways. Free arrestin-3 and receptor-bound arrestin-3 scaffold the ASK1-MKK4-JNK3 module, promoting JNK3 phosphorylation, whereas highly homologous arrestin-2 does not. Here, we used arrestin-2/3 chimeras and mutants to identify key residues of arrestin-3 responsible for its ability to facilitate JNK3 activation. Our data demonstrate that both arrestin domains are involved in JNK3 activation, with the C-terminal domain being more important than the N-terminal domain. We found that Val-343 is the key contributor to this function, whereas Leu-278, Ser-280, His-350, Asp-351, His-352, and Ile-353 play supporting roles. We also show that the arrestin-3-specific difference in the arrangement of the ?-strands in the C-terminal domain that underlies its lower selectivity for active phosphoreceptors does not play an appreciable role in its ability to enhance JNK3 activation. Importantly, the strength of the binding of ASK1 or JNK3, as revealed by the efficiency of co-immunoprecipitation, does not correlate with the ability of arrestin proteins to promote ASK1-dependent JNK3 phosphorylation. Thus, multiple residues on the non-receptor-binding side of arrestin-3 are crucial for JNK3 activation, and this function and the receptor-binding characteristics of arrestin can be manipulated independently by targeted mutagenesis.

SUBMITTER: Seo J

PROVIDER: S-EPMC3151035 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Identification of arrestin-3-specific residues necessary for JNK3 kinase activation.

Seo Jungwon J Tsakem Elviche L EL Breitman Maya M Gurevich Vsevolod V VV

The Journal of biological chemistry 20110629 32

Arrestins bind active phosphorylated G protein-coupled receptors, blocking G protein activation and channeling the signaling to G protein-independent pathways. Free arrestin-3 and receptor-bound arrestin-3 scaffold the ASK1-MKK4-JNK3 module, promoting JNK3 phosphorylation, whereas highly homologous arrestin-2 does not. Here, we used arrestin-2/3 chimeras and mutants to identify key residues of arrestin-3 responsible for its ability to facilitate JNK3 activation. Our data demonstrate that both ar ...[more]

PMID: 21715332

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Project description:PKR-like endoplasmic reticulum (ER) kinase (PERK) is an ER-associated stress sensor protein which phosphorylates eukaryotic initiation factor 2α (eIF2α) to induce translation attenuation in response to ER stress. PERK is also a regulator of lipogenesis during adipocyte differentiation through activation of the cleavage of sterol regulatory element binding protein 1 (SREBP1), resulting in the upregulation of lipogenic enzymes. Our recent studies have shown that human cytomegalovirus (HCMV) infection in human fibroblasts (HF) induces adipocyte-like lipogenesis through the activation of SREBP1. Here, we report that PERK expression is highly increased in HCMV-infected cells and is necessary for HCMV growth. Depletion of PERK, using short hairpin RNA (shRNA), resulted in attenuation of HCMV growth, inhibition of lipid synthesis and reduction of lipogenic gene expression. Examination of the cleavage of SREBP proteins showed PERK depletion inhibited the cleavage of SREBP1, but not SREBP2, in HCMV-infected cells, suggesting different cleavage regulatory mechanisms for SREBP1 and 2. Further studies showed that the depletion of SREBP1, but not SREBP2, reduced lipid synthesis in HCMV infection, suggesting that activation of SREBP1 is sufficient to induce lipogenesis in HCMV infection. The reduction of lipid synthesis by PERK depletion can be partially restored by expressing a Flag-tagged nuclear form of SREBP1a. Our studies also suggest that the induction of PERK in HCMV-infected cells stimulates SREBP1 cleavage by reducing levels of Insig1 (Insulin inducible gene 1) protein; this occurs independent of the phosphorylation of eIF2α. Introduction of an exogenous Insig1-Myc into HCMV infected cells significantly reduced HCMV growth and lipid synthesis. Our data demonstrate that the induction of PERK during HCMV infection is necessary for full activation of lipogenesis; this effect appears to be mediated by limiting the levels of Insig1 thus freeing SREBP1-SCAP complexes for SREBP1 processing.

Dataset Information

Identification of arrestin-3-specific residues necessary for JNK3 kinase activation.

Publications

Identification of arrestin-3-specific residues necessary for JNK3 kinase activation.

Similar Datasets

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