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  • Suppressor of IKKepsilon forms direct interactions with cytoskeletal proteins, tubulin and α-actinin, linking innate immunity to the cytoskeleton.

Suppressor of IKKepsilon forms direct interactions with cytoskeletal proteins, tubulin and α-actinin, linking innate immunity to the cytoskeleton.

FEBS open bio (2018-07-11)
Halie A Sonnenschein, Kenneth F Lawrence, Karli A Wittenberg, Frank A Slykas, Emerald L Dohleman, Jilan B Knoublauch, Sean M Fahey, Timothy M Marshall, James D Marion, Jessica K Bell
ABSTRACT

Suppressor of IKKepsilon (SIKE) is associated with the type I interferon response of the innate immune system through TANK-binding kinase 1 (TBK1). Originally characterized as an endogenous inhibitor of TBK1 when overexpressed in viral infection and pathological cardiac hypertrophic models, a mechanistic study revealed that SIKE acts as a high-affinity substrate of TBK1, but its function remains unknown. In this work, we report that scratch assay analysis of parental and SIKE CRISPR/Cas9 knockout HAP1 cells showed an ~ 20% decrease in cell migration. Investigation of the SIKE interaction network through affinity purification/mass spectrometry showed that SIKE formed interactions with cytoskeletal proteins. In immunofluorescence assays, endogenous SIKE localized to cytosolic puncta in both epithelial and myeloid cells and to nuclear puncta in myeloid cells, while in epithelial cells additional staining occurred in stress fiber-like structures and adjacent to the plasma membrane. Using cellular markers, co-occurrence of SIKE fluorescence with actin, α-actinin, and ezrin was detected. Reciprocal immunoprecipitation revealed a SIKE:tubulin interaction sensitive to the phosphorylation state of SIKE, but a SIKE:α-actinin interaction was unchanged by SIKE phosphorylation. In vitro precipitation assays confirmed a direct SIKE interaction with tubulin and α-actinin. These results indicate that SIKE may promote cell migration by directly associating with the cytoskeleton. In this role, SIKE may mediate cytoskeletal rearrangement necessary in innate immunity, but also link a key catalytic hub, TBK1, to the cytoskeleton. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD007262.

MATERIALS
Product Number
Brand
Product Description

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Monoclonal Anti-α-Actinin antibody produced in mouse, clone BM-75.2, ascites fluid
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Anti-SIKE1 antibody produced in rabbit, affinity isolated antibody, buffered aqueous glycerol solution, Ab2
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Monoclonal ANTI-FLAG® M2-Peroxidase (HRP) antibody produced in mouse, clone M2, purified immunoglobulin, buffered aqueous glycerol solution
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Anti-SIKE1 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution, Ab1