The PRMT5/HURP axis retards Golgi repositioning by stabilizing acetyl‐tubulin and Golgi apparatus during cell migration

Abstract

The Golgi apparatus (GA) translocates to the cell leading end during directional migration, thereby determining cell polarity and transporting essential factors to the migration apparatus. The study provides mechanistic insights into how GA repositioning (GR) is regulated. We show that the methyltransferase PRMT5 methylates the microtubule regulator HURP at R122. The HURP methylation mimicking mutant 122F impairs GR and cell migration. Mechanistic studies revealed that HURP 122F or endogenous methylated HURP, that is, HURP m122, interacts with acetyl-tubulin. Overexpression of HURP 122F stabilizes the bundling pattern of acetyl-tubulin by decreasing the sensitivity of the latter to a microtubule disrupting agent nocodazole. HURP 122F also rigidifies GA via desensitizing the organelle to several GA disrupting chemicals. Similarly, the acetyl-tubulin mimicking mutant 40Q or tubulin acetyltransferase αTAT1 can rigidify GA, impair GR, and retard cell migration. Reversal of HURP 122F-induced GA rigidification, by knocking down GA assembly factors such as GRASP65 or GM130, attenuates 122F-triggered GR and cell migration. Remarkably, PRMT5 is found downregulated and the level of HURP m122 is decreased during the early hours of wound healing-based cell migration, collectively implying that the PRMT5-HURP-acetyl-tubulin axis plays the role of brake, preventing GR and cell migration before cells reach empty space.

The JMJD6/HURP axis promotes cell migration via NF‐κB‐dependent centrosome repositioning and Cdc42‐mediated Golgi repositioning

“…To investigate the mechanisms by which JMJD6 regulates GR and cell migration, we noticed that JMJD6 is reported to act against PRMT5 by demethylating the substrates of PRMT5, and our previous works show that PRMT5 methylates HURP at R122 (Chiu et al, 2022). Therefore, we firstly tested the interaction of JMJD6 and HURP by performing immune‐coprecipitation and found that JMJD6 interacted with HURP, no matter exogenous or endogenous version of JMJD6 and HURP (Figure 2a).…”

“…The following antibodies, plasmids and reagents used in the study were listed below with working dilution or concentration, catalog number, and manufactory company: actin antibody (Western blot, WB ‐ 1:1000, sc8432, Santa cruz), α‐tubulin antibody (WB—1:1000; sc‐5286; Santa cruz), γ‐tubulin antibody (Immunofluorescence; IF—1:100; sc‐17788; Santa cruz), Cdc42 antibody (WB—1:1000; sc‐87; Santa cruz), GFP antibody (WB—1:2000; 11814460001; Roche), GRASP65 antibody (IF—1:1000; ab174834; Abcam), HA antibody (WB—1:2000; H3553; Sigma), HURP, HURP m122, and HURP nm122 antibody (Chiu et al, 2022), JMJD6 antibody (WB ‐ 1:500, sc‐32740, Santa cruz), NF‐кB antibody (IF ‐ 1:100, sc‐8008, Santa cruz), V5 antibody (WB ‐ 1:2000, GTX628592, Gene Tex), PADI4 antibody (WB—1:1000; #32493; Signalway), SAS6 antibody (WB—1:1000; sc376836; Santa Cruz), p EGFP (#13031; addgene), EGFP‐HURP WT, 122 K,122 F (Chiu et al, 2022), V5‐JMJD6 (B. Chang et al, 2007), p NF‐кB ‐Luc reporter plasmid (Chen et al, 2015), HA‐HURP (Yu et al, 2005), BFA (1 μg/ml; B6542, Sigma), ARF6 inhibitor SecinH3 (25 μM; CAS 853625‐60‐2; Santa Cruz), Cdc42 inhibitor ML141 (25 μM; SML0407; Sigma), NF‐кB inhibitor IV (50 μM; 481412; MERCK), PMA (1 μg/ml; P1585; Sigma), puromycin (2 μg/ml; ant‐pr‐5b; Invivogen). 293T and HeLa were from ATCC.…”

“…Our previous works show that the HURP m122 inactivates GR by triggering a GA intrinsic pathway. The PRMT5-induced HURP m122 interacts with and stabilizes GA-derived acetyl-tubulin, which in turn rigidifies GA structure and impairs GR and cell migration (Chiu et al, 2022). To examine the potential interplay of GA intrinsic inhibitory mechanism and the GA extrinsic activating signaling cascade involving NF-кB, CR, Cdc42, we reported that inactivation of Cdc42 blocked GR (Figure 3g), while did not significantly affect the structure or stability of GA (Figure 5g), implying that the GA extrinsic signaling factors such as Cdc42 may not affect GA intrinsic property including structure or rigidity of the organelle.…”

“…HURP is found localized to spindle (Tsou et al, 2003) and nucleus (Chen et al, 2015), and functions in assembling or stabilizing the mitotic microtubule spindle (Casanova et al, 2008;Silljé et al, 2006) and regulating the expression of cyclin E1 with the help of NF-κB (Chen et al, 2015). Our previous study shows that HURP is methylated by PRMT5 at R122, and the HURP m122 impairs cell migration via enhancing the stability of GA-derived acetyl-tubulin and then rigidifying GA structure (Chiu et al, 2022).…”

“…HURP is found localized to spindle (Tsou et al, 2003) and nucleus (Chen et al, 2015), and functions in assembling or stabilizing the mitotic microtubule spindle (Casanova et al, 2008; Silljé et al, 2006) and regulating the expression of cyclin E1 with the help of NF‐κB (Chen et al, 2015). Our previous study shows that HURP is methylated by PRMT5 at R122, and the HURP m122 impairs cell migration via enhancing the stability of GA‐derived acetyl‐tubulin and then rigidifying GA structure (Chiu et al, 2022). Since JMJD6 acts against PRMT5 by demethylating the substrates of PRMT5 (Tae et al, 2011; Tsai et al, 2017; Wu et al, 2015), and HURP is able to regulate cell migration (Q. Wang et al, 2018), the study focused itself on exploring the role of the JMJD6/HURP axis in the regulation of GR and cell migration.…”