Limits...
LIM domain kinases as potential therapeutic targets for neurofibromatosis type 2.

Petrilli A, Copik A, Posadas M, Chang LS, Welling DB, Giovannini M, Fernández-Valle C - Oncogene (2013)

Bottom Line: We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2(ΔEx2) MSCs in a dose-dependent manner, but did not affect viability of control MSCs.Similarly, LIMK knockdown decreased viability of Nf2(ΔEx2) MSCs.Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, College of Medicine, University of Central Florida, Orlando, FL, USA.

ABSTRACT
Neurofibromatosis type 2 (NF2) is caused by mutations in the NF2 gene that encodes a tumor-suppressor protein called merlin. NF2 is characterized by formation of multiple schwannomas, meningiomas and ependymomas. Merlin loss-of-function is associated with increased activity of Rac and p21-activated kinases (PAKs) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrate for Cdc42/Rac-PAK and modulate actin dynamics by phosphorylating cofilin at serine-3. This modification inactivates the actin severing and depolymerizing activity of cofilin. LIMKs also translocate into the nucleus and regulate cell cycle progression. Significantly, LIMKs are overexpressed in several tumor types, including skin, breast, lung, liver and prostate. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2(ΔEx2)) exhibited increased levels of LIMK1, LIMK2 and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared with wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared with normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2(ΔEx2) MSC reduced LIMK1 and LIMK2 levels. We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2(ΔEx2) MSCs in a dose-dependent manner, but did not affect viability of control MSCs. Similarly, LIMK knockdown decreased viability of Nf2(ΔEx2) MSCs. The decreased viability of Nf2(ΔEx2) MSCs was not due to caspase-dependent or -independent apoptosis, but rather due to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrests cells in early mitosis by decreasing aurora A activation. Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

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Inhibition of LIMKs decreases Aurora A autophosphorylation. (a) Representative western blot of three independent BMS-5 dose-response experiments. Nf2ΔEx2 MSCs were plated in half of a 12-well plate and treated the next day with increasing BMS-5 concentrations or DMSO for 8 hr. Cells were harvested, lysed and 10 µg of protein were analyzed by western blotting for phospho-Thr288-Aurora A and Aurora A. β-actin levels were used as loading controls. (b) Quantification of BMS-5 effect-response on Aurora A phosphorylation. Graph represents the mean ± SEM (n=3), **P<0.01; *** P<0.001 determined by one-way ANOVA using Dunnett’s multiple comparison test.
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Figure 7: Inhibition of LIMKs decreases Aurora A autophosphorylation. (a) Representative western blot of three independent BMS-5 dose-response experiments. Nf2ΔEx2 MSCs were plated in half of a 12-well plate and treated the next day with increasing BMS-5 concentrations or DMSO for 8 hr. Cells were harvested, lysed and 10 µg of protein were analyzed by western blotting for phospho-Thr288-Aurora A and Aurora A. β-actin levels were used as loading controls. (b) Quantification of BMS-5 effect-response on Aurora A phosphorylation. Graph represents the mean ± SEM (n=3), **P<0.01; *** P<0.001 determined by one-way ANOVA using Dunnett’s multiple comparison test.

Mentions: To identify additional down-stream effects of BMS-5 (beyond inhibition of cofilin-Ser3 phosphorylation) that could cause a G2/M arrest, we tested the levels of Aurora A phosphorylation. Aurora A interacts with both LIMK1 and 2 and is a major regulator of mitotic spindle assembly and chromosomal alignment and segregation. We found that Nf2ΔEx2 MSCs treated with increasing concentrations of BMS-5 had a dose dependent decrease phosphorylation of Aurora A at Thr288 (Figure 7 a, b).


LIM domain kinases as potential therapeutic targets for neurofibromatosis type 2.

Petrilli A, Copik A, Posadas M, Chang LS, Welling DB, Giovannini M, Fernández-Valle C - Oncogene (2013)

Inhibition of LIMKs decreases Aurora A autophosphorylation. (a) Representative western blot of three independent BMS-5 dose-response experiments. Nf2ΔEx2 MSCs were plated in half of a 12-well plate and treated the next day with increasing BMS-5 concentrations or DMSO for 8 hr. Cells were harvested, lysed and 10 µg of protein were analyzed by western blotting for phospho-Thr288-Aurora A and Aurora A. β-actin levels were used as loading controls. (b) Quantification of BMS-5 effect-response on Aurora A phosphorylation. Graph represents the mean ± SEM (n=3), **P<0.01; *** P<0.001 determined by one-way ANOVA using Dunnett’s multiple comparison test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4016185&req=5

Figure 7: Inhibition of LIMKs decreases Aurora A autophosphorylation. (a) Representative western blot of three independent BMS-5 dose-response experiments. Nf2ΔEx2 MSCs were plated in half of a 12-well plate and treated the next day with increasing BMS-5 concentrations or DMSO for 8 hr. Cells were harvested, lysed and 10 µg of protein were analyzed by western blotting for phospho-Thr288-Aurora A and Aurora A. β-actin levels were used as loading controls. (b) Quantification of BMS-5 effect-response on Aurora A phosphorylation. Graph represents the mean ± SEM (n=3), **P<0.01; *** P<0.001 determined by one-way ANOVA using Dunnett’s multiple comparison test.
Mentions: To identify additional down-stream effects of BMS-5 (beyond inhibition of cofilin-Ser3 phosphorylation) that could cause a G2/M arrest, we tested the levels of Aurora A phosphorylation. Aurora A interacts with both LIMK1 and 2 and is a major regulator of mitotic spindle assembly and chromosomal alignment and segregation. We found that Nf2ΔEx2 MSCs treated with increasing concentrations of BMS-5 had a dose dependent decrease phosphorylation of Aurora A at Thr288 (Figure 7 a, b).

Bottom Line: We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2(ΔEx2) MSCs in a dose-dependent manner, but did not affect viability of control MSCs.Similarly, LIMK knockdown decreased viability of Nf2(ΔEx2) MSCs.Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, College of Medicine, University of Central Florida, Orlando, FL, USA.

ABSTRACT
Neurofibromatosis type 2 (NF2) is caused by mutations in the NF2 gene that encodes a tumor-suppressor protein called merlin. NF2 is characterized by formation of multiple schwannomas, meningiomas and ependymomas. Merlin loss-of-function is associated with increased activity of Rac and p21-activated kinases (PAKs) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrate for Cdc42/Rac-PAK and modulate actin dynamics by phosphorylating cofilin at serine-3. This modification inactivates the actin severing and depolymerizing activity of cofilin. LIMKs also translocate into the nucleus and regulate cell cycle progression. Significantly, LIMKs are overexpressed in several tumor types, including skin, breast, lung, liver and prostate. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2(ΔEx2)) exhibited increased levels of LIMK1, LIMK2 and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared with wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared with normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2(ΔEx2) MSC reduced LIMK1 and LIMK2 levels. We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2(ΔEx2) MSCs in a dose-dependent manner, but did not affect viability of control MSCs. Similarly, LIMK knockdown decreased viability of Nf2(ΔEx2) MSCs. The decreased viability of Nf2(ΔEx2) MSCs was not due to caspase-dependent or -independent apoptosis, but rather due to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrests cells in early mitosis by decreasing aurora A activation. Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

Show MeSH
Related in: MedlinePlus