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.
Database
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 1 partner repository with the dataset identifier PXD007262.
SIKE is a high‐affinity substrate of TBK1, a kinase involved with innate immune sensing, but its function is unknown. We used CRISPR/Cas9 SIKE knockout cells to show that loss of SIKE reduced cell migration. Characterization of SIKE interactions revealed that phosphorylated SIKE forms interactions with the cytoskeletal proteins α‐actinin and tubulin. Altogether, these data support a role for SIKE linking innate immune sensing to cytoskeletal rearrangements.
Suppressor of
IKK
epsilon (
SIKE
) is associated with the type I interferon response of the innate immune system through
TANK
‐binding kinase 1 (
TBK
1). Originally characterized as an endogenous ...inhibitor of
TBK
1 when overexpressed in viral infection and pathological cardiac hypertrophic models, a mechanistic study revealed that
SIKE
acts as a high‐affinity substrate of
TBK
1, but its function remains unknown. In this work, we report that scratch assay analysis of parental and
SIKE CRISPR
/Cas9 knockout
HAP
1 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,
TBK
1, to the cytoskeleton.
Database
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the
PRIDE
1 partner repository with the dataset identifier
PXD
007262.
Abstract only
Innate immunity coordinates the first line of defense against invading pathogens. This involves the recruitment and subsequent migration of nearby immune cells to the site of infection, ...allowing the pathogen to be consumed via phagocytosis to sequester the infection. These processes require the rapid disassembly, rearrangement, and repolymerization of the network of structural proteins known as the cytoskeleton. Migration and phagocytosis are one of the functional consequences of innate immune receptor triggered signaling pathways. Signals coalesce at catalytic hubs, such as kinases, which serve to amplify and regulate the signal. In the anti‐viral response, TANK binding kinase 1 (TBK1) functions as a catalytic hub. Suppressor of IKK epsilon (SIKE) acts as a high affinity, alternate TBK1 substrate, although SIKE's downstream function is not yet defined. The primary goal of this work was to assess the effect of SIKE on cytoskeletal rearrangements in migration and phagocytosis. For these studies, a CRISPR knockout cell line was derived, SIKE‐CR, from chronic myelogenous leukemia (CML) cell line KBM‐7 named HAP1. Wound healing assays of HAP1 parental cells and SIKE‐CR illustrated that the absence of SIKE significantly decreased cell migration. In the mouse macrophage RAW 264.7 cell line, SIKE expression was knocked down via lentiviral delivery of shRNA targeting SIKE. Phagocytosis was assessed in WT and SIKE knockdown cells via uptake of latex beads labeled with IgG‐FITC. Reduced SIKE expression increased phagocytic uptake. To determine SIKE interactions with cytoskeletal proteins, co‐immunoprecipitations (cIP), in vitro precipitation (IVP) reactions, and enzyme‐linked immunosorbent assays (ELISA) were completed and demonstrated SIKE:tubulin and SIKE:α‐actinin complexes. Together, these studies establish an interaction between SIKE and cytoskeletal proteins and suggest that SIKE functions in cytoskeletal rearrangement associated with migration and phagocytosis.
Support or Funding Information
Work supported by NIH grant R21AI107447 and USD SURE.
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