Ito Yuki, Asada Akiko, Kobayashi Hiroyuki, Takano Tetsuya, Sharma Govinda, Saito Taro, Ohta Yasutaka, Amano Mutsuki, Kaibuchi Kozo, Hisanaga Shin-Ichi
Laboratory of Molecular Neuroscience, Department of Biological Sciences, Tokyo Metropolitan University, Minami-osawa, Hachioji, Tokyo 192-0397, Japan.
Division of Cell Biology, Department of Biosciences, School of Science, Kitasato University, 1-15-1 Kitasato, Minami, Sagamihara 252-0373, Japan.
Mol Cell Neurosci. 2014 Jul;61:34-45. doi: 10.1016/j.mcn.2014.05.006. Epub 2014 May 27.
Cdk5 is a member of the cyclin-dependent kinase (Cdk) family that plays a role in various neuronal activities including brain development, synaptic regulation, and neurodegeneration. Cdk5 requires the neuronal specific activators, p35 and p39 for subcellular compartmentalization. However, it is not known how active Cdk5 is recruited to F-actin cytoskeleton, which is a Cdk5 target. Here we found p35 and p39 localized to F-actin rich regions of the plasma membrane and investigated the underlying targeting mechanism in vitro by expressing them with Rho family GTPases in Neuro2A cells. Both p35 and p39 accumulated at the cell peripheral lamellipodia and perinuclear regions, where active Rac1 is localized. Interestingly, p35 and p39 displayed different localization patterns as p35 was found more at the perinuclear region and p39 was found more in peripheral lamellipodia. We then confirmed this distinct localization in primary hippocampal neurons. We also determined that the localization of p39 to lamellipodia requires myristoylation and Lys clusters within the N-terminal p10 region. Additionally, we found that p39-Cdk5, but not p35-Cdk5 suppressed lamellipodia formation by reducing Rac1 activity. These results suggest that p39-Cdk5 has a dominant role in Rac1-dependent lamellipodial activity.
细胞周期蛋白依赖性激酶5（Cdk5）是细胞周期蛋白依赖性激酶（Cdk）家族的成员，在包括大脑发育、突触调节和神经退行性变在内的各种神经元活动中发挥作用。Cdk5需要神经元特异性激活剂p35和p39进行亚细胞区室化。然而，目前尚不清楚活性Cdk5是如何被招募到作为Cdk5靶点的F-肌动蛋白细胞骨架上的。在这里，我们发现p35和p39定位于质膜富含F-肌动蛋白的区域，并通过在Neuro2A细胞中与Rho家族GTP酶一起表达来体外研究其潜在的靶向机制。p35和p39都聚集在细胞外周的片状伪足和核周区域，而活性Rac1就定位于这些区域。有趣的是，p35和p39表现出不同的定位模式，p35更多地出现在核周区域，而p39更多地出现在外周片状伪足中。然后我们在原代海马神经元中证实了这种独特的定位。我们还确定p39定位于片状伪足需要N端p10区域内的肉豆蔻酰化和赖氨酸簇。此外，我们发现p39-Cdk5，而不是p35-Cdk5，通过降低Rac1活性抑制片状伪足的形成。这些结果表明p39-Cdk5在Rac1依赖性片状伪足活动中起主导作用。
