Department of Biological Sciences, Tokyo Metropolitan University, Tokyo 192-0397, Japan.
J Neurosci. 2012 May 9;32(19):6587-99. doi: 10.1523/JNEUROSCI.5317-11.2012.
Axonal outgrowth is a coordinated process of cytoskeletal dynamics and membrane trafficking; however, little is known about proteins responsible for regulating the membrane supply. LMTK1 (lemur kinase 1)/AATYK1 (apoptosis-associated tyrosine kinase 1) is a serine/threonine kinase that is highly expressed in neurons. We recently reported that LMTK1 plays a role in recycling endosomal trafficking in CHO-K1 cells. Here we explore the role of LMTK1 in axonal outgrowth and its regulation by Cdk5 using mouse brain cortical neurons. LMTK1 was expressed and was phosphorylated at Ser34, the Cdk5 phosphorylation site, at the time of axonal outgrowth in culture and colocalized with Rab11A, the small GTPase that regulates recycling endosome traffic, at the perinuclear region and in the axon. Overexpression of the unphosphorylated mutant LMTK1-S34A dramatically promoted axonal outgrowth in cultured neurons. Enhanced axonal outgrowth was diminished by the inactivation of Rab11A, placing LMTK1 upstream of Rab11A. Unexpectedly, the downregulation of LMTK1 by knockdown or gene targeting also significantly enhanced axonal elongation. Rab11A-positive vesicles were transported anterogradely more quickly in the axons of LMTK1-deficient neurons than in those of wild-type neurons. The enhanced axonal outgrowth was reversed by LMTK1-WT or the LMTK1-S34D mutant, which mimics the phosphorylated state, but not by LMTK1-S34A. Thus, LMTK1 can negatively control axonal outgrowth by regulating Rab11A activity in a Cdk5-dependent manner, and Cdk5-LMTK1-Rab11 is a novel signaling pathway involved in axonal outgrowth.
轴突生长是细胞骨架动态和膜运输的协调过程；然而，对于负责调节膜供应的蛋白质知之甚少。LMTK1（狐猴激酶 1）/AATYK1（凋亡相关酪氨酸激酶 1）是一种在神经元中高度表达的丝氨酸/苏氨酸激酶。我们最近报道 LMTK1 在 CHO-K1 细胞中参与再循环内体运输。在这里，我们使用鼠脑皮质神经元探索 LMTK1 在轴突生长中的作用及其被 Cdk5 的调节。在培养物中轴突生长时，LMTK1 表达并在丝氨酸 34 处磷酸化，这是 Cdk5 的磷酸化位点，并且与 Rab11A（调节再循环内体运输的小 GTPase）在核周区域和轴突中共定位。过表达未磷酸化的突变体 LMTK1-S34A 可显著促进培养神经元中的轴突生长。通过 Rab11A 的失活，增强的轴突生长减弱，将 LMTK1 置于 Rab11A 的上游。出乎意料的是，通过敲低或基因靶向下调 LMTK1 也显著增强了轴突伸长。与野生型神经元相比，LMTK1 缺陷神经元中的 Rab11A 阳性囊泡在轴突中向前运输更快。增强的轴突生长可通过 LMTK1-WT 或模拟磷酸化状态的 LMTK1-S34D 突变体逆转，但不能通过 LMTK1-S34A 逆转。因此，LMTK1 可以通过 Cdk5 依赖性方式调节 Rab11A 活性来负性控制轴突生长，并且 Cdk5-LMTK1-Rab11 是参与轴突生长的新信号通路。
