Hyperphosphorylation of TAU and filopodial retraction following microinjection of protein kinase C catalytic subunits
Hyperphosphorylation of TAU and filopodial retraction following microinjection of protein kinase C catalytic subunits
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显微注射蛋白激酶 C 催化亚基后 TAU 过度磷酸化和丝状伪足回缩
DOI：
10.1002/jnr.490420507
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发表时间：
1995
影响因子：
4.2
通讯作者：
T. Shea
中科院分区：
文献类型：
--
作者：
Corrine M. Cressman;T. Shea
通过钙激活的蛋白水解对蛋白激酶C（PKC）的有限蛋白水解切割PKC的调节和催化亚基，产生游离的组成型激活的激酶（“PKM”），其与完整的亲本酶不同，不是钙依赖性的，并且不限于质膜。这些后者的性质留下开放的可能性，PKM可能有机会，因此可能磷酸化，基板通常不可用完整的PKC。我们通过将PKC和PKM，沿着罗丹明标记的葡聚糖示踪剂，显微注射到未分化的NB 2a/d 1小鼠神经母细胞瘤细胞中，研究了这种异常磷酸化在伴随阿尔茨海默病的神经变性的某些方面的潜在参与。4小时后，将培养物固定并用针对各种磷酸化状态的tau的单克隆抗体（PHF-1、ALZ-50、Tau-1、AT 8）进行免疫荧光处理，然后用荧光素缀合的二抗进行免疫荧光处理。在罗丹明照明下通过共注射的罗丹明缀合的葡聚糖示踪剂定位显微注射的细胞，之后在荧光素照明下检查抗体免疫反应性。显微光密度分析表明，PKC的显微注射并没有增加抗体的基础免疫荧光强度;相反，PKM的显微注射诱导PHF-1和ALZ-50水平分别增加了3倍和2倍。相比之下，在PKC或PKM显微注射后，在AT 8和Tau-1免疫荧光中没有观察到显著改变。虽然未分化的NB 2a/d1细胞通常会产生短的丝状伪足样神经突，但相差显微镜显示PKM注射细胞上没有丝状伪足或神经突，而PKC注射细胞与未注射细胞的丝状伪足样神经突的比例相似。无细胞分析证实了PKC在必要的辅因子存在下和PKM增加PHF-1和ALZ-50免疫反应性的能力;未观察到AT 8或Tau-1免疫反应性的变化。这些研究结果强调了有限的PKC蛋白水解产生PKM的异常扩增在某些病理条件下可能导致神经元变性的可能性。© 1995 Wiley利斯公司
Limited proteolysis of protein kinase C (PKC) by calcium‐activated proteolysis cleaves the regulatory and catalytic subunits of PKC, generating a free, constitutively activated kinase (“PKM”) that, unlike the intact parent enzyme, is not calcium‐dependent, and is not restricted to the plasma membrane. These latter properties leave open the possibility that PKM may have access to, and may therefore phosphorylate, substrates normally unavailable to intact PKC. We examined the potential involvement of such aberrant phosphorylation in certain aspects of the neurodegeneration accompanying Alzheimer's disease by microinjecting PKC and PKM, along with a rhodamine‐conjugated dextran tracer, into undifferentiated NB2a/d1 mouse neuroblastoma cells. After 4 hr, cultures were fixed and processed for immunofluorescence with monoclonal antibodies (PHF‐1, ALZ‐50, Tau‐1, AT8) directed against tau in various phosphorylation states followed by fluorescein‐conjugated secondary antibodies. Microinjected cells were localized via co‐injected rhodamine‐conjugated dextran tracer under rhodamine illumination, after which antibody immunoreactivity was examined under fluorescein illumination. Microdensitometric analyses indicated that microinjection of PKC did not increase basal immunofluorescent intensities of the antibodies; by contrast, microinjection of PKM induced threeand twofold increases in PHF‐1 and ALZ‐50 levels, respectively. By contrast, no significant alteration was observed in AT8 and Tau‐1 immunofluorescence following either PKC or PKM microinjection. Whereas undifferentiated NB2a/d1 cells typically elaborate short, filopodia‐like neurites, phase‐contrast microscopy revealed the absence of fllopodia or neurites on PKM‐injected cells, while a similar percentage of PKC‐injected cells elaborated filopodialike neurites as did uninjected cells. Cell‐free analyses confirmed the ability of PKC, in the presence of necessary co‐factors, and PKM to increase PHF‐1 and ALZ‐50 immunoreactivity; no change was observed in AT8 or Tau‐1 immunoreactivity. These findings underscore the possibility that an abnormal amplification in limited PKC proteolysis to generate PKM could, under certain pathological conditions, contribute to neuronal degeneration. © 1995 Wiley‐Liss, Inc.
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DOI：
10.1016/s0021-9258(18)48531-6
发表时间：
1992-01
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
S. G. Greenberg;P. Davies;J D Schein;L I Binder
通讯作者：
S. G. Greenberg;P. Davies;J D Schein;L I Binder
影响因子：
3.3
作者：
Thomas B. Shea;M. Beermann
通讯作者：
Thomas B. Shea;M. Beermann
DOI：
10.1016/s0021-9258(18)45420-8
发表时间：
1987-12
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Jacques BaudierS;-R.;David Cole
通讯作者：
Jacques BaudierS;-R.;David Cole
影响因子：
56.9
作者：
B. Wolozin;Alex Pruchnicki;D. Dickson;P. Davies
通讯作者：
B. Wolozin;Alex Pruchnicki;D. Dickson;P. Davies
在完整的人神经母细胞瘤细胞和无细胞条件下，蛋白激酶 C α 及其游离催化亚基蛋白激酶 M 的降解。
影响因子：
3.5
作者：
Shea,TB;Beermann,ML;Griffin,WR;Leli,U
通讯作者：
Leli,U