Abstract
Neurofibromin is a neurofibromatosis type 1 (NF1) tumor suppressor gene product with a domain that acts as a GTPase-activating protein and functions, in part, as a negative regulator of Ras. Loss of neurofibromin expression in NF1 patients is associated with elevated Ras activity and increased cell proliferation, predisposing to a variety of tumors of the peripheral and central nervous systems. We show here, using the small interfering RNA (siRNA) technique, that neurofibromin dynamically regulates actin cytoskeletal reorganization, followed by enhanced cell motility and gross cell aggregation in Matrigel matrix. NF1 siRNA induces characteristic morphological changes, such as excessive actin stress fiber formation, with elevated negative phosphorylation levels of cofilin, which regulates actin cytoskeletal reorganization by depolymerizing and severing actin filaments. We found that the elevated phosphorylation of cofilin in neurofibromin-depleted cells is promoted by activation of a Rho-ROCK-LIMK2 pathway, which requires Ras activation but is not transduced through three major Ras-mediated downstream pathways via Raf, phosphatidylinositol 3-kinase, and RalGEF. In addition, the exogenous expression of the NF1-GTPase-activating protein-related domain suppressed the NF1 siRNA-induced phenotypes. Neurofibromin was demonstrated to play a significant role in the machinery regulating cell proliferation and in actin cytoskeletal reorganization, which affects cell motility and adhesion. These findings may explain, in part, the mechanism of multiple neurofibroma formation in NF1 patients.
Highlights
Lisch nodules, and learning disability [2, 3]
We propose that neurofibromin plays a significant role in cell motility by regulating the dynamics and reorganization of actin filaments via the Rho-ROCK-LIM kinase 2 (LIMK2)-cofilin pathway
Immunocytochemical analysis showed that neurofibromatosis type 1 (NF1) small interfering RNA (siRNA) induced dynamic morphological changes according to the level of neurofibromin suppression with time
Summary
Learning disability [2, 3]. The hallmark of NF1 is benign tumors that develop in the peripheral nervous system accompanied by an increased risk of malignancies. Our results demonstrated that the acute knockdown of neurofibromin stimulates phosphorylation of cofilin by LIMK2 and induces characteristic morphological changes with abnormal actin stress fiber reorganization and formation of gross cell aggregates in matrix. Investigated various signal molecules that regulate actin cytoskeletal reorganization in the neurofibromin-depleted cells and found that phosphorylation of ADF/cofilin is significantly elevated with time in the cells, whereas their levels in control siRNA transfectants had no changes (Fig. 4A and supplemental Fig. S4).
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