Journal: Frontiers in Immunology

Article Title: Regulation of interferon alpha production by the MAGUK-family protein CASK under H5N1 infection

doi: 10.3389/fimmu.2024.1513713

Figure Lengend Snippet: HCK regulates CASK S395 phosphorylation and nuclear translocation via CDK5 during H5N1 infection. (A) There are 4 out of 9 Src-family kinase members express in myeloid cells. Its abundance in H5N1-infected GM-MΦ are quantified by RT-qPCR. (B) 293T cells were seeded onto coverslip and transfected with EGFP-CASK (either wild-type or S395A) and co-overexpressed with either pCMV-empty vector, pCMV-HCK-WT or pCMV-HCK-Y499F. 24 hours after transfection, cells were fixed and stained with anti-HCK antibody, anti-mouse APC conjugated antibody and Hoechst 33342, then analyzed with fluorescence confocal microscope. HCK-expressing cells are counted as either nuclear GFP positive or negative from 15 fields for each treatment group and the statistical analysis is shown in (C) . (D) Primary GM-MΦ were mock infected or infected with H5N1, MOI=1. Cells were seeded onto coverslip and treated with DMSO or CDK5 inhibitor 3 μM for 6 hours and then fixed, stained and analyzed by fluorescence confocal microscope. Nuclear CASK intensity from the images acquired in (D) is calculated by Las X software. (E, F) H5N1-infected GM-MΦ were treated with CDK5 inhibitor for 12 hours and culture supernatant were analyzed by IFNA2/4 ELISA (E) ; cells were lyzed with Trizol, total RNA were extracted and Ifna4 mRNA levels are analyzed by RT-qPCR (F) . *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t-test).

Article Snippet: For CASK staining, anti-CASK antibody (Sigma, SAB5200069) was used, while anti-HCK antibody (Santa Cruz, 101428) was employed for HCK staining.

Techniques: Phospho-proteomics, Translocation Assay, Infection, Quantitative RT-PCR, Transfection, Plasmid Preparation, Staining, Fluorescence, Microscopy, Expressing, Software, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Regulation of interferon alpha production by the MAGUK-family protein CASK under H5N1 infection

doi: 10.3389/fimmu.2024.1513713

Figure Lengend Snippet: Identification of HCK-dependent CASK phosphorylation site S395 by IP-LC-MS-MS.

Article Snippet: For CASK staining, anti-CASK antibody (Sigma, SAB5200069) was used, while anti-HCK antibody (Santa Cruz, 101428) was employed for HCK staining.

Techniques: Phospho-proteomics

Journal: Frontiers in Immunology

Article Title: Regulation of interferon alpha production by the MAGUK-family protein CASK under H5N1 infection

doi: 10.3389/fimmu.2024.1513713

Figure Lengend Snippet: Phosphorylated CASK enters nucleus and forms a complex to facilitate CRM1-mediated Ifna mRNP export during H5N1 infection When the H5N1 virus infects macrophages, PKR senses the 5’ tri-phosphate group of the viral genomic RNA, activating a signaling pathway that induces the expression of IFN-α. PKR activity is crucial for the transcriptional activation of Ifna , as treatment with a PKR inhibitor suppresses both CASK and IFN-α expression. During H5N1 infection, the expression and activity of HCK are upregulated, positively correlating with CDK5-mediated phosphorylation of CASK at S395. The S395-phosphorylated CASK translocates to the nucleus, where it forms a complex with STIP1, CCT4, and TNK1, as confirmed by IP-LC-MS/MS analysis. This CASK complex is recruited to the Ifna promoters through CCT4’s interaction with IRF7. Additionally, the complex associates with nascent Ifna mRNA via secondary interactions with HNRNPs and facilitates CRM1 recruitment to the transcript, enabling CRM1-mediated nuclear export. This Ifna mRNA-coupled CASK complex may counteract the competitive binding of influenza vRNP to CRM1, thereby supporting efficient nuclear export of Ifna mRNA.

Article Snippet: For CASK staining, anti-CASK antibody (Sigma, SAB5200069) was used, while anti-HCK antibody (Santa Cruz, 101428) was employed for HCK staining.

Techniques: Infection, Virus, Expressing, Activity Assay, Activation Assay, Phospho-proteomics, Liquid Chromatography with Mass Spectroscopy, Binding Assay

Journal: Frontiers in Immunology

Article Title: Regulation of interferon alpha production by the MAGUK-family protein CASK under H5N1 infection

doi: 10.3389/fimmu.2024.1513713

Figure Lengend Snippet: Antibodies for WB, IF, and IP.

Article Snippet: For CASK staining, anti-CASK antibody (Sigma, SAB5200069) was used, while anti-HCK antibody (Santa Cruz, 101428) was employed for HCK staining.

Techniques: