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Anti-migratory effect of vinflunine in endothelial and glioblastoma cells is associated with changes in EB1 C-terminal detyrosinated/tyrosinated status.

Rovini A, Gauthier G, Bergès R, Kruczynski A, Braguer D, Honoré S - PLoS ONE (2013)

Bottom Line: Such effect was associated with a reduction of EB1 comet length at microtubule (+) ends.Interestingly, we found a correlation between the reduction of EB1 comet length by vinflunine and the inhibition of cell migration.Using 3-L-Nitrotyrosine incorporation experiments, we concluded that the EB1 C-terminal modifications result from a detyrosination/retyrosination cycle as described for tubulin.

View Article: PubMed Central - PubMed

Affiliation: Aix Marseille Université, Institut National de la Santé et de la Recherche Médicale UMR_S 911, Marseille, France.

ABSTRACT
We previously showed that vinflunine, a microtubule-targeting drug of the Vinca-alkaloid family exerted its anti-angiogenic/anti-migratory activities through an increase in microtubule dynamics and an inhibition of microtubule targeting to adhesion sites. Such effect was associated with a reduction of EB1 comet length at microtubule (+) ends. In this work we first showed that the pro-angiogenic vascular endothelial growth factor VEGF suppressed microtubule dynamics in living Human Umbilical Vein Endothelial Cells (HUVECs), increased EB1 comet length by 40%, and induced EB1 to bind all along the microtubules, without modifying its expression level. Such microtubule (+) end stabilization occurred close to the plasma membrane in the vicinity of focal adhesion as shown by TIRF microscopy experiments. Vinflunine completely abolished the effect of VEGF on EB1 comets. Interestingly, we found a correlation between the reduction of EB1 comet length by vinflunine and the inhibition of cell migration. By using 2D gel electrophoresis we demonstrated for the first time that EB1 underwent several post-translational modifications in endothelial and tumor cells. Particularly, the C-terminal EEY sequence was poorly detectable in control and VEGF-treated HUVECs suggesting the existence of a non-tyrosinated form of EB1. By using specific antibodies that specifically recognized and discriminated the native tyrosinated form of EB1 and a putative C-terminal detyrosinated form, we showed that a detyrosinated form of EB1 exists in HUVECs and tumor cells. Interestingly, vinflunine decreased the level of the detyrosinated form and increased the native tyrosinated form of EB1. Using 3-L-Nitrotyrosine incorporation experiments, we concluded that the EB1 C-terminal modifications result from a detyrosination/retyrosination cycle as described for tubulin. Altogether, our results show that vinflunine inhibits endothelial cell migration through an alteration of EB1 comet length and EB1 detyrosination/retyrosination cycle.

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Detection of detyrosinated EB1 in HUVECs and U87 cells.(A) HUVECs incubation with 3-L-nitrotyrosine allowed detection, in total cell lysates, of a nitrotyrosinated form of tubulin but also of EB1. Representative of 3 independent experiments (B) Purified tyrosinated and detyrosinated EB1 (EB1-EEY, EB1ΔY) proteins were loaded as, respectively, negative and positive probes in regard to detyrosinated EB1 detection in HUVECS and U87 total cell lysate by using a specific guinea pig anti-detyrosinated EB1 antibody [22] (C) Transwell migration assay was performed in U87 cells depleted of EB1 (clone sh11) and expressing GFP-EB1 full length or GFP-EB1ΔY mutant. U87 sh0 clone was also used as a negative control. Data show the average number of cells that migrated. At least three independent experiments were performed for each condition. Bar ± S.E.M. (*) indicates significant differences from controls: *, p<0.05; **, p<0.005. (D) Western blot was performed with down-regulating EB1 U87 sh11 clone transfected or not with GFP-EB1 full length, GFP-EB1ΔY expression plasmids or control GFP-empty vector. U87 sh0 clone was also used as a negative control. Tyrosinated-EB1 was detected by using a specific anti-EB1 tyrosinated antibody [22].
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pone-0065694-g003: Detection of detyrosinated EB1 in HUVECs and U87 cells.(A) HUVECs incubation with 3-L-nitrotyrosine allowed detection, in total cell lysates, of a nitrotyrosinated form of tubulin but also of EB1. Representative of 3 independent experiments (B) Purified tyrosinated and detyrosinated EB1 (EB1-EEY, EB1ΔY) proteins were loaded as, respectively, negative and positive probes in regard to detyrosinated EB1 detection in HUVECS and U87 total cell lysate by using a specific guinea pig anti-detyrosinated EB1 antibody [22] (C) Transwell migration assay was performed in U87 cells depleted of EB1 (clone sh11) and expressing GFP-EB1 full length or GFP-EB1ΔY mutant. U87 sh0 clone was also used as a negative control. Data show the average number of cells that migrated. At least three independent experiments were performed for each condition. Bar ± S.E.M. (*) indicates significant differences from controls: *, p<0.05; **, p<0.005. (D) Western blot was performed with down-regulating EB1 U87 sh11 clone transfected or not with GFP-EB1 full length, GFP-EB1ΔY expression plasmids or control GFP-empty vector. U87 sh0 clone was also used as a negative control. Tyrosinated-EB1 was detected by using a specific anti-EB1 tyrosinated antibody [22].

Mentions: According to the requirement of the last tyrosine residue for EB1/CAP-Gly interaction [29] and the similarity in the C-terminal –EEY sequence between α-tubulin and EB1, one can expect to find the same post-translational modifications to be generated and notably a cycle of detyrosination/retyrosination. HUVECs incubation with 3-L-nitrotyrosine, a non-genetically coded amino acid, during 48 h resulted in its incorporation in tubulin, as expected (Figure 3A) [31], [32]. Interestingly, we found that EB1 also incorporated 3-L-nitrotyrosine (Figure 3A). As this result led to suspect the existence of a detyrosinated form of EB1, we tested its existence in both endothelial and glioblastoma cells by using a specific antibody against the detyrosinated form of EB1 [22]. This antibody that failed to recognize purified tyrosinated EB1 (Figure 3B), detected a detyrosinated form of EB1 in HUVEC and U87 cell lysates (Figure 3B).


Anti-migratory effect of vinflunine in endothelial and glioblastoma cells is associated with changes in EB1 C-terminal detyrosinated/tyrosinated status.

Rovini A, Gauthier G, Bergès R, Kruczynski A, Braguer D, Honoré S - PLoS ONE (2013)

Detection of detyrosinated EB1 in HUVECs and U87 cells.(A) HUVECs incubation with 3-L-nitrotyrosine allowed detection, in total cell lysates, of a nitrotyrosinated form of tubulin but also of EB1. Representative of 3 independent experiments (B) Purified tyrosinated and detyrosinated EB1 (EB1-EEY, EB1ΔY) proteins were loaded as, respectively, negative and positive probes in regard to detyrosinated EB1 detection in HUVECS and U87 total cell lysate by using a specific guinea pig anti-detyrosinated EB1 antibody [22] (C) Transwell migration assay was performed in U87 cells depleted of EB1 (clone sh11) and expressing GFP-EB1 full length or GFP-EB1ΔY mutant. U87 sh0 clone was also used as a negative control. Data show the average number of cells that migrated. At least three independent experiments were performed for each condition. Bar ± S.E.M. (*) indicates significant differences from controls: *, p<0.05; **, p<0.005. (D) Western blot was performed with down-regulating EB1 U87 sh11 clone transfected or not with GFP-EB1 full length, GFP-EB1ΔY expression plasmids or control GFP-empty vector. U87 sh0 clone was also used as a negative control. Tyrosinated-EB1 was detected by using a specific anti-EB1 tyrosinated antibody [22].
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Related In: Results  -  Collection

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pone-0065694-g003: Detection of detyrosinated EB1 in HUVECs and U87 cells.(A) HUVECs incubation with 3-L-nitrotyrosine allowed detection, in total cell lysates, of a nitrotyrosinated form of tubulin but also of EB1. Representative of 3 independent experiments (B) Purified tyrosinated and detyrosinated EB1 (EB1-EEY, EB1ΔY) proteins were loaded as, respectively, negative and positive probes in regard to detyrosinated EB1 detection in HUVECS and U87 total cell lysate by using a specific guinea pig anti-detyrosinated EB1 antibody [22] (C) Transwell migration assay was performed in U87 cells depleted of EB1 (clone sh11) and expressing GFP-EB1 full length or GFP-EB1ΔY mutant. U87 sh0 clone was also used as a negative control. Data show the average number of cells that migrated. At least three independent experiments were performed for each condition. Bar ± S.E.M. (*) indicates significant differences from controls: *, p<0.05; **, p<0.005. (D) Western blot was performed with down-regulating EB1 U87 sh11 clone transfected or not with GFP-EB1 full length, GFP-EB1ΔY expression plasmids or control GFP-empty vector. U87 sh0 clone was also used as a negative control. Tyrosinated-EB1 was detected by using a specific anti-EB1 tyrosinated antibody [22].
Mentions: According to the requirement of the last tyrosine residue for EB1/CAP-Gly interaction [29] and the similarity in the C-terminal –EEY sequence between α-tubulin and EB1, one can expect to find the same post-translational modifications to be generated and notably a cycle of detyrosination/retyrosination. HUVECs incubation with 3-L-nitrotyrosine, a non-genetically coded amino acid, during 48 h resulted in its incorporation in tubulin, as expected (Figure 3A) [31], [32]. Interestingly, we found that EB1 also incorporated 3-L-nitrotyrosine (Figure 3A). As this result led to suspect the existence of a detyrosinated form of EB1, we tested its existence in both endothelial and glioblastoma cells by using a specific antibody against the detyrosinated form of EB1 [22]. This antibody that failed to recognize purified tyrosinated EB1 (Figure 3B), detected a detyrosinated form of EB1 in HUVEC and U87 cell lysates (Figure 3B).

Bottom Line: Such effect was associated with a reduction of EB1 comet length at microtubule (+) ends.Interestingly, we found a correlation between the reduction of EB1 comet length by vinflunine and the inhibition of cell migration.Using 3-L-Nitrotyrosine incorporation experiments, we concluded that the EB1 C-terminal modifications result from a detyrosination/retyrosination cycle as described for tubulin.

View Article: PubMed Central - PubMed

Affiliation: Aix Marseille Université, Institut National de la Santé et de la Recherche Médicale UMR_S 911, Marseille, France.

ABSTRACT
We previously showed that vinflunine, a microtubule-targeting drug of the Vinca-alkaloid family exerted its anti-angiogenic/anti-migratory activities through an increase in microtubule dynamics and an inhibition of microtubule targeting to adhesion sites. Such effect was associated with a reduction of EB1 comet length at microtubule (+) ends. In this work we first showed that the pro-angiogenic vascular endothelial growth factor VEGF suppressed microtubule dynamics in living Human Umbilical Vein Endothelial Cells (HUVECs), increased EB1 comet length by 40%, and induced EB1 to bind all along the microtubules, without modifying its expression level. Such microtubule (+) end stabilization occurred close to the plasma membrane in the vicinity of focal adhesion as shown by TIRF microscopy experiments. Vinflunine completely abolished the effect of VEGF on EB1 comets. Interestingly, we found a correlation between the reduction of EB1 comet length by vinflunine and the inhibition of cell migration. By using 2D gel electrophoresis we demonstrated for the first time that EB1 underwent several post-translational modifications in endothelial and tumor cells. Particularly, the C-terminal EEY sequence was poorly detectable in control and VEGF-treated HUVECs suggesting the existence of a non-tyrosinated form of EB1. By using specific antibodies that specifically recognized and discriminated the native tyrosinated form of EB1 and a putative C-terminal detyrosinated form, we showed that a detyrosinated form of EB1 exists in HUVECs and tumor cells. Interestingly, vinflunine decreased the level of the detyrosinated form and increased the native tyrosinated form of EB1. Using 3-L-Nitrotyrosine incorporation experiments, we concluded that the EB1 C-terminal modifications result from a detyrosination/retyrosination cycle as described for tubulin. Altogether, our results show that vinflunine inhibits endothelial cell migration through an alteration of EB1 comet length and EB1 detyrosination/retyrosination cycle.

Show MeSH
Related in: MedlinePlus