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The NF2 tumor suppressor regulates microtubule-based vesicle trafficking via a novel Rac, MLK and p38(SAPK) pathway.

Hennigan RF, Moon CA, Parysek LM, Monk KR, Morfini G, Berth S, Brady S, Ratner N - Oncogene (2012)

Bottom Line: Conversely, an activated Rac mutant decreased Rab6 vesicle velocity.Vesicle motility assays in isolated squid axoplasm further demonstrated that both mutant merlin and active Rac specifically reduce anterograde microtubule-based transport of vesicles dependent upon the activity of p38(SAPK) kinase.Taken together, our data suggest loss of merlin results in the Rac-dependent decrease of anterograde trafficking of exocytic vesicles, representing a possible mechanism controlling the concentration of growth factor receptors at the cell surface.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA. Robert.Hennigan@cchmc.org

ABSTRACT
Neurofibromatosis type 2 patients develop schwannomas, meningiomas and ependymomas resulting from mutations in the tumor suppressor gene, NF2, encoding a membrane-cytoskeleton adapter protein called merlin. Merlin regulates contact inhibition of growth and controls the availability of growth factor receptors at the cell surface. We tested if microtubule-based vesicular trafficking might be a mechanism by which merlin acts. We found that schwannoma cells, containing merlin mutations and constitutive activation of the Rho/Rac family of GTPases, had decreased intracellular vesicular trafficking relative to normal human Schwann cells. In Nf2-/- mouse Schwann (SC4) cells, re-expression of merlin as well as inhibition of Rac or its effector kinases, MLK and p38(SAPK), each increased the velocity of Rab6 positive exocytic vesicles. Conversely, an activated Rac mutant decreased Rab6 vesicle velocity. Vesicle motility assays in isolated squid axoplasm further demonstrated that both mutant merlin and active Rac specifically reduce anterograde microtubule-based transport of vesicles dependent upon the activity of p38(SAPK) kinase. Taken together, our data suggest loss of merlin results in the Rac-dependent decrease of anterograde trafficking of exocytic vesicles, representing a possible mechanism controlling the concentration of growth factor receptors at the cell surface.

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Merlin Inhibits Anterograde Axonal Transport in Isolated Squid AxoplasmMembrane bound organelle velocities were measured after perfusion of axoplasm with buffer supplemented with 0.5 to 1 μM purified recombinant merlin, or recombinant merlin plus the p38SAPK inhibitor, SB203580. Anterograde (blue) and retrograde (red) velocity measurements taken between 25 and 45 minutes were plotted as points. Curves were fitted for anterograde movement and retrograde movement using an exponential curve fit. A) Full length, wild-type merlin. B) C-terminal tail domain deletion mutant, FERM-Helix. C) FERM-Helix plus SB203580. D) The phosphorylation site mutant S518A. E) The phosphomimetic mutant S518D. F) The S518D mutant plus SB203580.
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Figure 6: Merlin Inhibits Anterograde Axonal Transport in Isolated Squid AxoplasmMembrane bound organelle velocities were measured after perfusion of axoplasm with buffer supplemented with 0.5 to 1 μM purified recombinant merlin, or recombinant merlin plus the p38SAPK inhibitor, SB203580. Anterograde (blue) and retrograde (red) velocity measurements taken between 25 and 45 minutes were plotted as points. Curves were fitted for anterograde movement and retrograde movement using an exponential curve fit. A) Full length, wild-type merlin. B) C-terminal tail domain deletion mutant, FERM-Helix. C) FERM-Helix plus SB203580. D) The phosphorylation site mutant S518A. E) The phosphomimetic mutant S518D. F) The S518D mutant plus SB203580.

Mentions: Perfusion of squid axoplasm with 1 μM purified recombinant wild type merlin protein had little or no inhibitory effect on the velocity of membrane bound organelles in the anterograde direction over the 50 minute assay period (Figure 6A). We then tested FERM-Helix, a C-terminal deletion mutant of merlin containing amino acids 1-502, that was shown to be FERM accessible relative to wild type by virtue of increased interaction with NHERF(7). Perfusion with the FERM-Helix mutant significantly reduced anterograde vesicle velocity (Figure 6B). Inhibition of p38SAPK activity by co-perfusion with the SB203580 inhibitor rescued anterograde vesicle inhibition by the FERM-Helix mutant (Figure 6C). Perfusing a nonphosphorylatable mutant, S518A, had little or no effect on vesicle velocity in this system (Figure 6D), a similar response to wild type. A phosphomimetic mutant S518D, which is predicted to have an accessible FERM domain and fails to suppress cell growth (13), also reduced anterograde vesicle transport (Figure 6E). This effect was not rescued by SB203580, suggesting different mechanisms of inhibition between FERM-Helix and S518D (Figure 6F). Retrograde velocity was essentially unchanged in all proteins tested. These data suggest that the open, FERM accessible isoforms of merlin, FERM-Helix and S518D can specifically regulate kinesin mediated anterograde vesicle transport. This inhibition occurs in the cell-free squid axoplasm system and is thus independent of the plasma membrane, where the bulk of merlin is localized, and of the cell nucleus, another reported site of merlin action (37). The dependence of FERM-Helix inhibition of anterograde vesicle transport on p38SAPK activity indicates that suppression is mediated by a kinase chain initiated by merlin, leading to a reduction in anterograde vesicle velocity.


The NF2 tumor suppressor regulates microtubule-based vesicle trafficking via a novel Rac, MLK and p38(SAPK) pathway.

Hennigan RF, Moon CA, Parysek LM, Monk KR, Morfini G, Berth S, Brady S, Ratner N - Oncogene (2012)

Merlin Inhibits Anterograde Axonal Transport in Isolated Squid AxoplasmMembrane bound organelle velocities were measured after perfusion of axoplasm with buffer supplemented with 0.5 to 1 μM purified recombinant merlin, or recombinant merlin plus the p38SAPK inhibitor, SB203580. Anterograde (blue) and retrograde (red) velocity measurements taken between 25 and 45 minutes were plotted as points. Curves were fitted for anterograde movement and retrograde movement using an exponential curve fit. A) Full length, wild-type merlin. B) C-terminal tail domain deletion mutant, FERM-Helix. C) FERM-Helix plus SB203580. D) The phosphorylation site mutant S518A. E) The phosphomimetic mutant S518D. F) The S518D mutant plus SB203580.
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Related In: Results  -  Collection

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Figure 6: Merlin Inhibits Anterograde Axonal Transport in Isolated Squid AxoplasmMembrane bound organelle velocities were measured after perfusion of axoplasm with buffer supplemented with 0.5 to 1 μM purified recombinant merlin, or recombinant merlin plus the p38SAPK inhibitor, SB203580. Anterograde (blue) and retrograde (red) velocity measurements taken between 25 and 45 minutes were plotted as points. Curves were fitted for anterograde movement and retrograde movement using an exponential curve fit. A) Full length, wild-type merlin. B) C-terminal tail domain deletion mutant, FERM-Helix. C) FERM-Helix plus SB203580. D) The phosphorylation site mutant S518A. E) The phosphomimetic mutant S518D. F) The S518D mutant plus SB203580.
Mentions: Perfusion of squid axoplasm with 1 μM purified recombinant wild type merlin protein had little or no inhibitory effect on the velocity of membrane bound organelles in the anterograde direction over the 50 minute assay period (Figure 6A). We then tested FERM-Helix, a C-terminal deletion mutant of merlin containing amino acids 1-502, that was shown to be FERM accessible relative to wild type by virtue of increased interaction with NHERF(7). Perfusion with the FERM-Helix mutant significantly reduced anterograde vesicle velocity (Figure 6B). Inhibition of p38SAPK activity by co-perfusion with the SB203580 inhibitor rescued anterograde vesicle inhibition by the FERM-Helix mutant (Figure 6C). Perfusing a nonphosphorylatable mutant, S518A, had little or no effect on vesicle velocity in this system (Figure 6D), a similar response to wild type. A phosphomimetic mutant S518D, which is predicted to have an accessible FERM domain and fails to suppress cell growth (13), also reduced anterograde vesicle transport (Figure 6E). This effect was not rescued by SB203580, suggesting different mechanisms of inhibition between FERM-Helix and S518D (Figure 6F). Retrograde velocity was essentially unchanged in all proteins tested. These data suggest that the open, FERM accessible isoforms of merlin, FERM-Helix and S518D can specifically regulate kinesin mediated anterograde vesicle transport. This inhibition occurs in the cell-free squid axoplasm system and is thus independent of the plasma membrane, where the bulk of merlin is localized, and of the cell nucleus, another reported site of merlin action (37). The dependence of FERM-Helix inhibition of anterograde vesicle transport on p38SAPK activity indicates that suppression is mediated by a kinase chain initiated by merlin, leading to a reduction in anterograde vesicle velocity.

Bottom Line: Conversely, an activated Rac mutant decreased Rab6 vesicle velocity.Vesicle motility assays in isolated squid axoplasm further demonstrated that both mutant merlin and active Rac specifically reduce anterograde microtubule-based transport of vesicles dependent upon the activity of p38(SAPK) kinase.Taken together, our data suggest loss of merlin results in the Rac-dependent decrease of anterograde trafficking of exocytic vesicles, representing a possible mechanism controlling the concentration of growth factor receptors at the cell surface.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA. Robert.Hennigan@cchmc.org

ABSTRACT
Neurofibromatosis type 2 patients develop schwannomas, meningiomas and ependymomas resulting from mutations in the tumor suppressor gene, NF2, encoding a membrane-cytoskeleton adapter protein called merlin. Merlin regulates contact inhibition of growth and controls the availability of growth factor receptors at the cell surface. We tested if microtubule-based vesicular trafficking might be a mechanism by which merlin acts. We found that schwannoma cells, containing merlin mutations and constitutive activation of the Rho/Rac family of GTPases, had decreased intracellular vesicular trafficking relative to normal human Schwann cells. In Nf2-/- mouse Schwann (SC4) cells, re-expression of merlin as well as inhibition of Rac or its effector kinases, MLK and p38(SAPK), each increased the velocity of Rab6 positive exocytic vesicles. Conversely, an activated Rac mutant decreased Rab6 vesicle velocity. Vesicle motility assays in isolated squid axoplasm further demonstrated that both mutant merlin and active Rac specifically reduce anterograde microtubule-based transport of vesicles dependent upon the activity of p38(SAPK) kinase. Taken together, our data suggest loss of merlin results in the Rac-dependent decrease of anterograde trafficking of exocytic vesicles, representing a possible mechanism controlling the concentration of growth factor receptors at the cell surface.

Show MeSH
Related in: MedlinePlus