Rosales Jesusa L, Ernst Joel D, Hallows Janice, Lee Ki-Young
Department of Cell Biology and Anatomy, Cancer Biology and Neuroscience Research Groups, The University of Calgary, Calgary, Alberta T2N 4N1, Canada.
J Biol Chem. 2004 Dec 24;279(52):53932-6. doi: 10.1074/jbc.M408467200. Epub 2004 Oct 18.
We have previously shown evidence for the existence of a calcium-independent, GTP-regulated mechanism of secretion from neutrophils, but this secretory mechanism remains to be fully elucidated. Cyclin-dependent kinase 5 (Cdk5), the various substrates of which include Munc18 and synapsin 1, has been implicated in neuronal secretion. Although the Cdk5 activator, p35, and Cdk5-p35 activity are primarily associated with neurons, we report here that p35 also exists in neutrophils and that an active Cdk5-p35 complex is present in these cells. Cdk5-p35 activity in human neutrophils is mostly localized in secretory granules, which show an increase in Cdk5-p35 level and activity upon GTP stimulation. The potent Cdk5 inhibitor, roscovitine, completely blocks GTP-stimulated granule Cdk5 activity, which accompanies lactoferrin secretion from neutrophil-specific granules. Roscovitine also inhibits GTP-induced lactoferrin secretion and surface localization of the secretion markers, CD63 and CD66b, to a certain extent. Furthermore, neutrophils from wild-type mice treated with roscovitine and neutrophils from p35(-/-) mice exhibit comparable surface expression levels of both CD63 and CD66b upon GTP stimulation. Although our data suggest that other molecules control GTP-induced secretion from neutrophils, it is clear that Cdk5-p35 is required to elicit the maximum GTP-induced secretory response. Our observation that multiple proteins in neutrophil granules serve as specific substrates of Cdk5 further supports the premise that the kinase is a key component of the GTP-regulated secretory apparatus in neutrophils.
我们之前已证明存在一种不依赖钙、受GTP调节的中性粒细胞分泌机制，但这种分泌机制仍有待充分阐明。细胞周期蛋白依赖性激酶5（Cdk5）的各种底物包括Munc18和突触素1，它与神经元分泌有关。尽管Cdk5激活剂p35和Cdk5 - p35活性主要与神经元相关，但我们在此报告p35也存在于中性粒细胞中，并且这些细胞中存在活性Cdk5 - p35复合物。人类中性粒细胞中的Cdk5 - p35活性大多定位于分泌颗粒，在GTP刺激后，这些颗粒中Cdk5 - p35的水平和活性会增加。强效Cdk5抑制剂roscovitine完全阻断GTP刺激的颗粒Cdk5活性，这与乳铁蛋白从嗜中性粒细胞特异性颗粒中分泌相伴。Roscovitine在一定程度上也抑制GTP诱导的乳铁蛋白分泌以及分泌标志物CD63和CD66b的表面定位。此外，用roscovitine处理的野生型小鼠的中性粒细胞和p35(-/-)小鼠的中性粒细胞在GTP刺激后，CD63和CD66b的表面表达水平相当。虽然我们的数据表明其他分子控制GTP诱导的中性粒细胞分泌，但很明显Cdk5 - p35是引发最大GTP诱导分泌反应所必需的。我们观察到中性粒细胞颗粒中的多种蛋白质作为Cdk5的特异性底物，这进一步支持了该激酶是中性粒细胞中GTP调节分泌装置关键组成部分的前提。
