CN103160569A - 调节细胞微丝动态新机制的发现 - Google Patents
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Abstract
神经细胞是一种由轴突，树突和细胞体构成的重要极性细胞。轴突的形成是神经细胞分化形成的形态指标。细胞骨架的动态变化是轴突形成所必须的。由肌动蛋白聚合而成的微丝是重要的细胞骨架之一，其动态变化的分子机理还不清楚。本研究首次发现，从秀丽线虫到哺乳动物具有保守性的UNC-51蛋白激酶既能结合又能磷酸化肌动蛋白，而且能使微丝形成颗粒状分布并输送颗粒状微丝到细胞质基质中去；UNC-51蛋白激酶还能把N1E115神经瘤细胞诱导变成分叉神经状细胞；无激酶活性的UNC-51突变体(K39M)蛋白则与微丝一起在细胞核周围形成积聚体构造物，不能诱导N1E115细胞成为分叉神经状细胞。根据以上发现结果，提出以下模型：UNC-51蛋白激酶通过直接地磷酸化肌动蛋白和输送颗粒状微丝到细胞质基质中的分子机理来调节细胞骨架的动态变化，进而调节神经细胞轴突的形成和功能。
Description
技术领域：
本发明属于基础生命科学，生物化学，细胞生物学，分子生物学，脑科学，肌肉科学，神经细胞骨架学和神经细胞信号转导学等多个领域，创新性地揭示了细胞微丝动态变化的新机理和神经细胞形成及工作的最新原理。
背景技术：
细胞微丝的动态和神经细胞轴突及肌肉细胞的形成和功能，细胞分裂以及细胞运动等有密切的关系。通常认为，细胞内微丝的形成和功能间接地被微丝结合蛋白(成核蛋白Arp2/3复合物，丝切蛋白cofilin等)的磷酸化影响，但微丝动态的基本机理仍然不清楚。还没有关于既直接磷酸化肌动蛋白也以其磷酸化机理影响微丝动态的激酶的报道。模式生物秀丽线虫和哺乳动物的神经系统在形态学和生物化学上是相似的；一个保存性基因在秀丽线虫和哺乳动物的不同实验系里能表达相同的功效。秀丽线虫的unc-51基因编码一个从线虫到人类的进化上具有保存性的丝/苏氨酸蛋白激酶，是神经形成所必须的；这个基因的突变引起线虫的神经形成及运动的欠缺和短粗体形。揭示unc-51基因所编码的蛋白激酶的底物和其生物化学功能的意义是重要的。
发明内容：
本发明的目的在于揭示unc-51基因所编码的蛋白激酶的重要底物和其生物化学功能，弄清楚神经细胞形成的分子机理，为神经和肌肉疾病的治疗提供崭新的原理和方法。本发明的内容为：
(1)首次揭示肌动蛋白既能和UNC-51激酶结合，更能作为此激酶的底物被磷酸化
肌动蛋白和UNC-51激酶的结合如图1，图2和图5所示。图1是把表达UNC-51激酶蛋白的人胚肾HEKs293细胞的溶胞产物用于免疫印迹后的结果，这图显示在被转染的HEKs293细胞内，UNC-51激酶蛋白结合着肌动蛋白。图2的体外酶联免疫吸附测定结果显示，来自秀丽线虫及兔肌的肌动蛋白都能够结合UNC-51激酶蛋白。图5是激光扫描荧光共焦显微镜的图像，这图像显示在非州绿猴肾细胞(COS-7)质基质中，带红色荧光的颗粒状UNC-51蛋白激酶和带绿色萤光的颗粒状肌动蛋白结合产生了颗粒状的黄色荧光。
图3显示UNC-51激酶在体外能够磷酸化来自兔肌的肌动蛋白并自我磷酸化，ATP和GTP 均可作为磷酸供体。图4显示秀丽线虫和兔肌的肌动蛋白的丝氨酸被UNC-51激酶磷酸化。
(2)首次揭示UNC-51蛋白激酶活性能输送颗粒状微丝到细胞质基质中去
和图6中无激酶活性的UNC-51突变蛋白(K39M)在细胞核周围形成的积聚体相比，图5显示，过量表达UNC-51激酶蛋白(红色)的COS-7细胞中，UNC-51激酶活性能输送粒状肌动蛋白到细胞质基质中去。
(3)首次揭示UNC-51蛋白激酶活性能够诱导成神经细胞瘤N1E115细胞变成多分枝的神经性细胞
图7显示，UNC-51激酶活性能诱导成神经细胞瘤N1E115细胞成为分叉神经似的细胞。
图8表示UNC-51激酶活性通过对细胞内肌动蛋白的磷酸化和运送颗粒状肌动蛋白到细胞质基质中，诱导神经轴突形成的模型。
附图说明：
图1肌动蛋白和UNC-51激酶的体内结合
图2肌动蛋白和UNC-51激酶的体外结合
图3UNC-51激酶在体外磷酸化肌动蛋白并自我磷酸化
图4UNC-51激酶在体外磷酸化秀丽线虫和兔肌的肌动蛋白的丝氨酸
图5UNC-51激酶活性能输送粒状肌动蛋白到细胞质基质中去
图6无激酶活性的UNC-51突变蛋白(K39M)和肌动蛋白在细胞核周围形成积聚体
图7UNC-51激酶活性能诱导成神经细胞瘤N1E115细胞成为分叉神经似的细胞
图8UNC-51激酶活性通过对细胞内肌动蛋白的磷酸化和输送粒状肌动蛋白诱导神经轴突形成的模型
具体实施方式：
【1】UNC-51蛋白激酶和肌动蛋白结合的测定
用细胞溶解缓冲液溶解表达UNC-51蛋白激酶的HEK293细胞，得到的溶胞产物的上清用于免疫印迹。利用亲和层析法从以上溶胞产物的上清纯化得到UNC-51蛋白激酶。运用固相酶联免疫测定法(ELISA)分析肌动蛋白和UNC-51蛋白激酶的体外结合。
【2】激酶活性的测定
在含有50μM末端放射性表记的腺苷三磷酸[γ-32P]-ATP或者鸟苷三磷酸[γ-32P]-GTP的Hepes激酶缓冲反应液(pH7.4)中，进行了UNC-51蛋白激酶磷酸化肌动蛋白的反应。用6.5％的SDS-PAGE和X-线胶片放射自显影法分析测定了激酶活性。薄层纤维素板(TLC)上的双向 分离电泳法用于磷酸化丝氨酸的同定。
【3】激光扫描荧光共焦显微镜的观察
DAPI用于细胞核染色，TexasRed和FITC分别用于UNC-51和肌动蛋白的染色。ZeissLSM510共焦显微镜被用于细胞观察和记录。
Claims (3)
- 本发明首次发现了UNC-51的激酶活性能够直接地磷酸化肌动蛋白并和细胞微丝一起以粒状构造分布到细胞质基质。UNC-51是第一个既磷酸化肌动蛋白也直接地影响微丝构造和分布的激酶。其关键发现为：1.在试管内的生化实验中，UNC-51的激酶活性直接地磷酸化非肌肉细胞肌动蛋白，肌肉细胞肌动蛋白和秀丽线虫肌动蛋白，在试管内和在活体内的生化实验中，UNC-51结合非肌肉细胞肌动蛋白，肌肉细胞肌动蛋白或秀丽线虫肌动蛋白。
- 2.在活体内的生化实验中，UNC-51和细胞微丝一起成为粒状构造和运输粒状构造微丝到细胞质基质。纤维状微丝+UNC-51+ATP(GTP)→磷酸化粒状微丝+ADP(GDP)
- 3.上记二项要求的技术原理在生命科学，脑科学，肿瘤科学，计算机科学，宇宙科学，交通科学，医学，药学和制药学，中药科学，各种疾病的治疗法，医疗技术以及所有其他方面的应用和推广。
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| CN1311199A (zh) * | 2000-02-29 | 2001-09-05 | 复旦大学 | 一种新的多肽——人肌动蛋白细丝蛋白21和编码这种多肽的多核苷酸 |
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