Figures & data
Figure 1. Cyclin dependent kinase 5 (CDK5) suppresses the production of IFN-β.
Note: A. A549 cells were stimulated with VSV-GFP (MOI = 0.1) for 1, 2, 4, 6 or 8 hours, and the phosphorylated or total proteins were detected with specific antibodies.
B-C. After transfection with empty vector (EV) or Flag-CDK5, A549 cells were treated for 10 hours with VSV-GFP (MOI = 0.1), followed by qRT-PCR analysis for detecting the RNA levels of viral genome (B) and the transcription level of VSV P protein (C).
D. TCID50 of VSV in the supernatant of A549 cells overexpressing Flag-CDK5 or EV was measured after infection with VSV-GFP (MOI = 0.1) for 10 hours.
E. EV or Flag-CDK5 was overexpressed in A549 cells, followed by infection with VSV-GFP (MOI = 0.1) for 4 or 8 hours, qRT-PCR analysis (upper panels) and immunoblotting assay (lower panels).
F. A549 cells overexpressing Flag-CDK5 or EV were stimulated with VSV-GFP (MOI = 0.1) for 4 or 8 hours, and IFN-β in supernatant was quantified using the ELISA kit. Lower panels represent immunoblotting assays.
G-H. Left panel: A549 cells overexpressing Flag-CDK5 or EV were treated with VSV-GFP (MOI = 0.1) for 4 or 8 hours, and the changes in protein phosphorylation were detected by Western blotting with specific antibodies. Right panel: Quantitative analysis of the Western blotting results. The relative abundances of P-TBK1, P-IRF3 or P-P65 were normalized to that of total TBK1, IRF3 or P65 (G), and the relative abundances of P-STAT1 or P-STAT2 were normalized to that of total STAT1 or STAT2 (H). All graph values are presented as means ± SE, n = 3. *P < 0.05, **P < 0.01.
Figure 2. CDK5 deficiency promotes IFN-β production.
Note: A. The mRNA expression of IFN-β in A549 cells that stably expressing short hairpin RNA specific to CDK5 (shCDK5) or negative control (shNC). After infection with VSV-GFP (MOI = 0.1) for 4 or 8 hours, qRT-PCR analysis (upper panels) and immunoblotting assay (lower panels) were conducted.
B. shNC and shCDK5 A549 cells were stimulated with VSV-GFP (MOI = 0.1) for 4 or 8 hours, and IFN-β in supernatant was quantified using the ELISA kit.
C-D. Left panel: shNC and shCDK5 A549 cells were treated with VSV-GFP (MOI = 0.1) for 2, 4 or 6 hours, the changes in protein phosphorylation were detected by Western blotting with specific antibodies. Right panel: Quantitative analysis of the Western blotting results. The relative abundances of P-TBK1, P-IRF3 or P-P65 were normalized to that of total TBK1, IRF3 or P65 (G), and the relative abundances of P-STAT1 or P-STAT2 were normalized to that of total STAT1 or STAT2 (H).
E-F. shNC and shCDK5 A549 cells were treated with VSV-GFP (MOI = 0.1) for 10 hours, followed by qRT-PCR analysis for detecting the RNA levels of viral genome (E) and the transcription level of VSV P protein (F).
G. TCID50 of VSV in the supernatant of shNC and shCDK5 A549 cells was measured after infection with VSV-GFP (MOI = 0.1) for 10 hours.
H. After stable knockdown of CDK5, A549 cells were transfected with V5-tagged CDK5 resistant plasmid, followed by 8 hours of VSV-GFP (MOI = 0.1) infection. The mRNA expression of IFN-β was quantified by qRT-PCR after RNA extraction. Right panels represent immunoblotting assays.
All graph values are presented as means ± SE, n = 3. *P < 0.05, **P < 0.01.
Figure 3. The regulation of CDK5 in IFN-β production is independent of its kinase activity.
Note: A. Flag-CDK5 was co-transfected with P35-V5 into 293T cells. After infection with VSV-GFP for 2, 4 or 6 hours, the cells were lysed and anti-Flag beads were used for immunoprecipitation.
B. After 8 hours of VSV-GFP (MOI = 0.1) infection, the mRNA expression of IFN-β in A549 cells overexpressing Flag-CDK5, Flag-CDK5 kinase inactive mutant (Flag-mutant) or EV was analysed by qRT-PCR assay
C. Left panel: Flag-CDK5, Flag-mutant or EV plasmids were transfected into A549 cells, followed by 8-hour infection with VSV-GFP (MOI = 0.1). Western blotting was performed to determine the phosphorylated or total protein of IRF3. Right panel: Quantitative analysis of the relative abundances of P-IRF3 normalized to that of total IRF3.
D-E. A549 cells were pre-treated with CDK5 kinase inhibitor Roscovitine (ROSC) for 30 minutes, followed by VSV-GFP (MOI = 0.1) infection for 8 hours with ROSC. Western blotting was performed to determine the phosphorylated or total protein of IRF3 (D), and IFN-β expression was analysed by qRT-PCR assay (E).
All graph values are presented as means ± SE, n = 3. *P < 0.05, **P < 0.01.
Figure 4. CDK5 attenuates the expression of retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5).
Note: A-B. Left panel: The expression levels of RIG-I and MDA5 were detected in A549 cells with EV and Flag-CDK5 overexpression (A) or NC and CDK5 knockdown (B) after VSV-GFP (MOI = 0.1) infection for 2, 4 or 6 hours. Right panel: Quantitative analysis of the relative abundances of RIG-I and MDA5 normalized to that of β-Actin in CDK5 overexpressing cells (A) or in CDK5-deficient cells (B).
C-D. Flag-RIG-I was co-transfected with increasing doses of CDK5-GFP into HEK293T cells (C) or A549 cells (D). After 24 hours of transfection, the expression levels of Flag-RIG-I and CDK5-GFP were determined by Western blotting.
E-F. Flag-MDA5 was co-transfected with increasing doses of CDK5-GFP into HEK293T cells (E) or A549 cells (F). After 24 hours of transfection, the protein levels of Flag-MDA5 and CDK5-GFP were detected by Western blotting.
G. HEK293T cells were transfected with Flag-RIG-I and EV or CDK5-GFP for 24 hours, followed by treatment with 3-MA (10 mM), NH4Cl (10 mM) or MG132 (20 μM) for 8 hours. The expression levels of Flag-RIG-I and CDK5-GFP were analysed by Western blotting.
H. HEK293T cells were co-transfected Flag-MDA5 with EV or CDK5-GFP for 24 hours, followed by treatment with 3-MA (10 mM), NH4Cl (10 mM) or MG132 (20 μM) for 8 hours. Western blotting demonstrated the expression levels of Flag-MDA5 and CDK5-GFP.
I-J. A549 cells overexpressing Flag-CDK5 or EV were infected with VSV-GFP (MOI = 0.1) for 8 hours. Total RNA was extracted, and the mRNA expression levels of RIG-I (I) and MDA5 (J) were analysed by qRT-PCR assays. Lower panels represent immunoblotting assays.
All graph values are presented as means ± SE, n = 3. **P < 0.01.
Figure 5. CDK5 is associated with myeloid differentiation primary response gene 88 (MyD88) and suppresses TLR-MyD88-mediated signalling pathways.
Note: A. Identification of CDK5-associated protein via mass spectrometry.
B. HEK293T cells were transfected with MyD88-Flag, and MyD88-Flag was pulled down from lysate by anti-Flag beads for Western blotting analysis.
C. Domain mapping of MyD88 and CDK5 interaction was performed by transfecting CDK5-V5 with different domain of MyD88-Flag into HEK293T cells. Anti-Flag beads were mixed into cell lysates for Western blotting analysis.
D. HEK293T cells overexpressing CDK5-GFP and EV or Myd88-Flag plasmids were infected with VSV-GFP (MOI = 0.1) for 3 or 6 hours. Cells were lysed, and CDK5-GFP was pulled down by anti-GFP beads for Western blotting analysis.
E-G. Effects of CDK5 on TLR2 (E), TLR4 (F) and TLR7 (G) signalling pathways. shNC and shCDK5 A549 cells were stimulated with zymosan (100 ng/ml) (E), lipopolysaccharide (LPS) (100 ng/ml) (F) or imiquimod (IMQ) (50 ng/ml) (G) for 2 hours, followed by qRT-PCR analysis (left panels) and immunoblotting assay (right panels).
All graph values are presented as means ± SE, n ≥ 3. *P < 0.05, **P < 0.01.
Figure 6. CDK5 negatively regulates the formation of MyD88 homodimers independent of its kinase activity.
Note: A. Endogenous expression patterns of MyD88 in A549 cells transfected with increasing doses of CDK5.
B. CDK5 attenuated the formation of MyD88 homodimers. MyD88-GFP was co-transfected with EV or CDK5-HA into HEK293T for 24 hours. After 3 hours of VSV-GFP (MOI = 0.1) infection, the cells were crosslinked by 0.1 mM BS3 for 30 minutes. The cell lysates were prepared after terminating the crosslink reaction for immunoblotting analysis. The expression of CDK5-HA was detected from the sample collected before cross-linkage. The right panel displayed the quantitative analysis of the relative abundances of MyD88 homodimers, which were crosslinked with BS3 and normalized to the monomer formation of MyD88.
C. Left panel: CDK5 deficiency promoted the formation of MyD88 homodimer. shNC and shCDK5 HEK293T cells were transfected with MyD88-GFP. The cells were then crosslinked by 0.1 mM BS3 for 30 minutes after 3 hours of VSV-GFP (MOI = 0.1) infection. The cell lysates were prepared after terminating the crosslink reaction for immunoblotting analysis. The right panel displayed the quantitative analysis of the relative abundances of MyD88 homodimers, which were crosslinked with BS3 and normalized to the monomer formation of MyD88.
D. Left panel: The formation of MyD88 homodimers was attenuated by CDK5 kinase inactive mutant. MyD88-GFP was co-transfected with EV, CDK5-HA WT (CDK5-HA) or CDK5-HA kinase dead mutant (mutant-HA) into HEK293T for 24 hours. The cells were crosslinked for 30 minutes with 0.1 mM BS3. The cell lysates were prepared after terminating the crosslink reaction for immunoblotting analysis. The right panel displays the quantitative analysis of the relative abundances of MyD88 homodimers, which were crosslinked with BS3 and normalized to the monomer formation of MyD88.
E. The self-interaction of MyD88 was attenuated by CDK5 or its kinase inactive mutant. After transfection with the indicated plasmids for 24 hours, MyD88-GFP in HEK293T cells was immunoprecipitated by anti-GFP beads for Western blotting analysis.
F. CDK5 suppressed the self-association of MyD88 by disturbing both the DD and TIR domains of MyD88. After transfection with the indicated plasmids for 24 hours, MyD88-GFP in HEK293T cells was immunoprecipitated by anti-GFP beads for Western blotting analysis.
G. Model of CDK5 in the regulation of IFN-β production triggered by RNA virus via TLR-MyD88 pathway.
All graph values are presented as means ± SE, n = 3.
Supplemental material
Supplemental Material
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The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materialshttps://doi.org/10.6084/m9.figshare.23512494.