Figure 7.
TBK1 phosphorylation of NAP1 at S318 impacts its stability during mitosis. (A and B) Representative immunoblots of asynchronous, G2, mitotic, and cytokinetic DLD-1 cells with or without TBK1 inhibitor treatment (MRT67307) for 1 h prior to G2 and throughout release timepoints. Asynchronous cells were treated for 2 h. Cells were synchronized at G2 using RO-3306. G2 cells were released for ∼30–45 min to collect mitotic samples and for ∼80–90 min to collect cytokinetic cells. (B) Semiquantitative analysis of A normalizing NAP1 protein levels to vinculin in asynchronous and mitotic conditions with or without MRT67307. Error bars indicate ± SEM; n = 3 independent experiments. (C) Phos-Tag gel analysis of NAP1 mitotic protein from DLD-1 cells either with MRT67307 for 1 h prior to G2 and throughout release or with 1 h phosphatase treatment. 6% SDS-PAGE gel was run in tandem to ensure equal protein loading. (D) Representative immunoblots of asynchronous, G2, mitotic, and cytokinetic cells from WT HeLa and stable TBK1 K38A rescue lines. Cells were synchronized at G2 using RO-3306. G2 cells were released for ∼30–45 min to collect mitotic samples and for ∼80–90 min to collect cytokinetic cells. (E) Semiquantitative analysis of (D) normalizing NAP1 protein levels to vinculin in asynchronous and mitotic conditions in WT HeLa and stable TBK1 K38A rescue lines. Error bars indicate ± SEM; n = 3 independent experiments. (F) Phos-Tag gel analysis of NAP1 mitotic protein from HeLa, K38A TBK1 rescue, and HeLa cells treated 1 h with phosphatase. 6% SDS-PAGE gel was run in tandem to ensure equal protein loading. (G) Representatives immunoblot analysis of NAP1 in asynchronous and mitotic cells from stable NAP1 and NAP1 S318A rescue lines. Cells were synchronized using RO-3306. (H) Semiquantitative analysis of (G) normalizing NAP1 protein levels in asynchronous and mitotic conditions in stable NAP1 rescue and stable NAP1 S318A rescue lines. Error bars indicate ± SEM; n = 3 independent experiments. (I) NAP1 activates TBK1 on centrosomes by binding to its adaptor binding C′ domain. Activated TBK1 phosphorylates NAP1 on S318 which acts as a signal for ubiquitin proteasomal degradation of NAP1. Loss of either of these centrosomal proteins, NAP1 or TBK1, impairs mitosis and cytokinesis leading to retention of multinucleated cells. One dot = one independent experiment. One-way ANOVA was performed for all statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001.

TBK1 phosphorylation of NAP1 at S318 impacts its stability during mitosis. (A and B) Representative immunoblots of asynchronous, G2, mitotic, and cytokinetic DLD-1 cells with or without TBK1 inhibitor treatment (MRT67307) for 1 h prior to G2 and throughout release timepoints. Asynchronous cells were treated for 2 h. Cells were synchronized at G2 using RO-3306. G2 cells were released for ∼30–45 min to collect mitotic samples and for ∼80–90 min to collect cytokinetic cells. (B) Semiquantitative analysis of A normalizing NAP1 protein levels to vinculin in asynchronous and mitotic conditions with or without MRT67307. Error bars indicate ± SEM; n = 3 independent experiments. (C) Phos-Tag gel analysis of NAP1 mitotic protein from DLD-1 cells either with MRT67307 for 1 h prior to G2 and throughout release or with 1 h phosphatase treatment. 6% SDS-PAGE gel was run in tandem to ensure equal protein loading. (D) Representative immunoblots of asynchronous, G2, mitotic, and cytokinetic cells from WT HeLa and stable TBK1 K38A rescue lines. Cells were synchronized at G2 using RO-3306. G2 cells were released for ∼30–45 min to collect mitotic samples and for ∼80–90 min to collect cytokinetic cells. (E) Semiquantitative analysis of (D) normalizing NAP1 protein levels to vinculin in asynchronous and mitotic conditions in WT HeLa and stable TBK1 K38A rescue lines. Error bars indicate ± SEM; n = 3 independent experiments. (F) Phos-Tag gel analysis of NAP1 mitotic protein from HeLa, K38A TBK1 rescue, and HeLa cells treated 1 h with phosphatase. 6% SDS-PAGE gel was run in tandem to ensure equal protein loading. (G) Representatives immunoblot analysis of NAP1 in asynchronous and mitotic cells from stable NAP1 and NAP1 S318A rescue lines. Cells were synchronized using RO-3306. (H) Semiquantitative analysis of (G) normalizing NAP1 protein levels in asynchronous and mitotic conditions in stable NAP1 rescue and stable NAP1 S318A rescue lines. Error bars indicate ± SEM; n = 3 independent experiments. (I) NAP1 activates TBK1 on centrosomes by binding to its adaptor binding C′ domain. Activated TBK1 phosphorylates NAP1 on S318 which acts as a signal for ubiquitin proteasomal degradation of NAP1. Loss of either of these centrosomal proteins, NAP1 or TBK1, impairs mitosis and cytokinesis leading to retention of multinucleated cells. One dot = one independent experiment. One-way ANOVA was performed for all statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal