Ohnishi H, Yamamori S, Ono K, Aoyagi K, Kondo S, Takahashi M
Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan.
Proc Natl Acad Sci U S A. 2001 Sep 11;98(19):10930-5. doi: 10.1073/pnas.191368198. Epub 2001 Sep 4.
Tyrosine kinases are expressed in many tissues, particularly in the central nervous system, and regulate various cellular functions. We report here that a src family tyrosine kinase-specific inhibitor, PP2, enhances neurotransmitter release from PC12 cells and primary cultured neurons. PP2 enhances only Ca(2+)-dependent release; it does not affect basal release. These effects result from an enhancement of vesicular exocytosis and not from the reuptake or refilling of neurotransmitters because Ca(2+)-dependent secretion of an exogenously expressed reporter protein, the human growth hormone (hGH), is also enhanced by PP2. Overexpression of constitutive active v-src, but not of a kinase-inactive mutant, suppressed Ca(2+)-dependent release. In PP2-treated cells, Pyk2, paxillin, and some other proteins showed a decrease in tyrosine phosphorylation, and the enhancement of tyrosine phosphorylation of these proteins in response to Ca(2+) influx was also reduced. Electron and fluorescence microscopy showed that PP2 treatment induced morphological change and decreased phalloidin reactivity at the filopodium-like structures on the processes of PC12 cells. Interestingly, inhibition of actin polymerization with cytochalasin D and latrunculin A enhanced Ca(2+)-dependent, but not basal, release. It is possible that a src family tyrosine kinase, through the regulation of actin dynamics, has an inhibitory function to regulate neurotransmitter release.
酪氨酸激酶在许多组织中表达，尤其是在中枢神经系统中，并调节各种细胞功能。我们在此报告，一种src家族酪氨酸激酶特异性抑制剂PP2可增强PC12细胞和原代培养神经元的神经递质释放。PP2仅增强钙依赖性释放；它不影响基础释放。这些作用是由于囊泡胞吐作用增强，而非神经递质的再摄取或再填充，因为PP2也增强了外源性表达的报告蛋白人生长激素（hGH）的钙依赖性分泌。组成型活性v-src的过表达而非激酶失活突变体的过表达抑制了钙依赖性释放。在PP2处理的细胞中，Pyk2、桩蛋白和其他一些蛋白质的酪氨酸磷酸化水平降低，并且这些蛋白质对钙内流的酪氨酸磷酸化增强也减少。电子显微镜和荧光显微镜显示，PP2处理诱导了形态变化，并降低了PC12细胞突起上丝状伪足样结构处的鬼笔环肽反应性。有趣的是，用细胞松弛素D和拉春库林A抑制肌动蛋白聚合可增强钙依赖性释放，但不增强基础释放。src家族酪氨酸激酶可能通过调节肌动蛋白动力学对神经递质释放具有抑制作用。
