Yamauchi Junji, Miyamoto Yuki, Kusakawa Shinji, Torii Tomohiro, Mizutani Reiko, Sanbe Atsushi, Nakajima Hideki, Kiyokawa Nobutaka, Tanoue Akito
Department of Pharmacology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535, Japan.
Exp Cell Res. 2008 Jul 1;314(11-12):2279-88. doi: 10.1016/j.yexcr.2008.03.019. Epub 2008 Apr 8.
Valproic acid (VPA), the drug for bipolar disorder and epilepsy, has a potent ability to induce neuronal differentiation, yet comparatively little is presently known about the underlying mechanism. We previously demonstrated that c-Jun N-terminal kinase (JNK) phosphorylation of the focal adhesion protein paxillin mediates differentiation in N1E-115 neuroblastoma cells. Here, we show that VPA up-regulates the neurofibromatosis type 2 (NF2) tumor suppressor, merlin, to regulate neurite outgrowth through the interaction with paxillin. The inhibition of merlin function by its knockdown or expression of merlin harboring the Gln-538-to-Pro mutation, a naturally occurring NF2 missense mutation deficient in linking merlin to the actin cytoskeleton, decreases VPA-induced neurite outgrowth. Importantly, the expression of merlin by itself is not sufficient to induce neurite outgrowth, which requires co-expression with paxillin, the binding partner of merlin. In fact, the missense mutation Trp-60-to-Cys or Phe-62-to-Ser, that is deficient in binding to paxillin, reduces neurite outgrowth induced by VPA. In addition, co-expression of a paxillin construct harboring the mutation at the JNK phosphorylation site with merlin results in blunted induction of the outgrowth. We also find that the first LIM domain of paxillin is a major binding region with merlin and that expression of the isolated first LIM domain blocks the effects of VPA. Furthermore, similar findings that merlin regulates neurite outgrowth through the interaction with paxillin have been observed in several kinds of neuronal cells. These results suggest that merlin is an as yet unknown regulator of neurite outgrowth through the interaction with paxillin, providing a possibly common mechanism regulating neurite formation.
丙戊酸（VPA）是用于治疗双相情感障碍和癫痫的药物，具有强大的诱导神经元分化的能力，但目前对其潜在机制了解相对较少。我们之前证明，粘着斑蛋白桩蛋白的c-Jun氨基末端激酶（JNK）磷酸化介导了N1E-115神经母细胞瘤细胞的分化。在此，我们表明VPA上调2型神经纤维瘤病（NF2）肿瘤抑制因子默林，通过与桩蛋白相互作用来调节神经突生长。通过敲低默林功能或表达携带Gln-538-to-Pro突变的默林（一种自然发生的NF2错义突变，缺乏将默林与肌动蛋白细胞骨架连接的能力）来抑制默林功能，会减少VPA诱导的神经突生长。重要的是，默林自身的表达不足以诱导神经突生长，这需要与默林的结合伴侣桩蛋白共表达。事实上，缺乏与桩蛋白结合能力的错义突变Trp-60-to-Cys或Phe-62-to-Ser会减少VPA诱导的神经突生长。此外，在JNK磷酸化位点携带突变的桩蛋白构建体与默林共表达会导致生长诱导减弱。我们还发现桩蛋白的第一个LIM结构域是与默林的主要结合区域，分离的第一个LIM结构域的表达会阻断VPA的作用。此外，在几种神经元细胞中也观察到了默林通过与桩蛋白相互作用来调节神经突生长的类似发现。这些结果表明，默林是一种尚未被发现的通过与桩蛋白相互作用来调节神经突生长的调节因子，为调节神经突形成提供了一种可能的共同机制。
