Abstract
In vertebrate species, the innate immune system down-regulates protein translation in response to viral infection through the action of the double-stranded RNA (dsRNA)-activated protein kinase (PKR). In some teleost species another protein kinase, Z-DNA-dependent protein kinase (PKZ), plays a similar role but instead of dsRNA binding domains, PKZ has Zα domains. These domains recognize the left-handed conformer of dsDNA and dsRNA known as Z-DNA/Z-RNA. Cyprinid herpesvirus 3 infects common and koi carp, which have PKZ, and encodes the ORF112 protein that itself bears a Zα domain, a putative competitive inhibitor of PKZ. Here we present the crystal structure of ORF112-Zα in complex with an 18-bp CpG DNA repeat, at 1.5 Å. We demonstrate that the bound DNA is in the left-handed conformation and identify key interactions for the specificity of ORF112. Localization of ORF112 protein in stress granules induced in Cyprinid herpesvirus 3-infected fish cells suggests a functional behavior similar to that of Zα domains of the interferon-regulated, nucleic acid surveillance proteins ADAR1 and DAI.
Highlights
§ To whom correspondence should be addressed: Endocrine-Hypertension Div., Dept. of Medicine, Brigham and Women’s Hospital and Harvard Medical School, 221 Longwood Ave., Boston, MA 02115
With the use of the yeast two-hybrid system, we identified filamin-A (an actin-cross-linking, putative scaffold protein that binds mitogen-activated protein kinase (MAPK) components activated by the calcium-sensing receptor (CaR)) as an intracellular binding partner of the CaR’s carboxyl (COOH)-terminal tail
Introducing the portion of filamin-A interacting with the CaR into CaR-transfected HEK-293 cells using protein transduction with a His-tagged, Tat-filamin-A fusion protein nearly abolished CaR-mediated activation of ERK1/2 MAPK but had no effect on ERK1/2 activity stimulated by ADP
Summary
Ca2ϩo, extracellular calcium concentration; CaR, calcium-sensing receptor; GST, glutathione S-transferase; HEK-293, human embryonic kidney 293; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; NTA, nitrilotriacetic acid. Domains 14 and 15 of filamin-A, which is a large actin-binding, putative scaffold protein that binds caveolin-1 [13, 14], a key structural protein of caveolae [11], as well as several components of MAPK signaling cascades (e.g. mitogen-activated protein kinase/extracellular signal-regulated kinase kinases 1/2 and stress-activated protein kinase/ERK kinase-1/mitogenactivated protein kinase/extracellular signal-regulated kinase kinase kinase (Ref. 15)). Interfering with the CaR-filamin-A interaction nearly abrogates CaR- but not ADP-mediated activation of ERK1/2, suggesting that the specific interaction between the CaR and filamin-A could play an important role in the CaR’s coupling to activation of MAPK
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