Phosphorylation of vimentin in mitotically selected cells. In vitro cyclic AMP-independent kinase and calcium-stimulated phosphatase activities.
Phosphorylation of vimentin in mitotically selected cells. In vitro cyclic AMP-independent kinase and calcium-stimulated phosphatase activities.
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丝分裂选择细胞中波形蛋白的磷酸化。体外环状AMP独立的激酶和钙刺激的磷酸酶活性。
DOI：
10.1083/jcb.108.1.67
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发表时间：
1989-01
影响因子：
7.8
通讯作者：
Evans, R M
中科院分区：
文献类型：
--
作者：
Evans, R M
在体外条件下检测了中间丝蛋白vimentin的磷酸化。非有丝分裂和有丝分裂选择小鼠L-929细胞的细胞质和triton不溶性细胞骨架制剂显示出明显不依赖于cAMP和Ca2+的波形蛋白激酶活性。从有丝分裂选择细胞制备的蛋白激酶活性水平高于非有丝分裂细胞。在有丝分裂细胞质中添加Ca2+降低了净维门蛋白磷酸化。去磷酸化实验表明，这些制剂中存在磷酸酶活性，这是由Ca2+的添加刺激的。对DEAE-Sephacel和磷酸纤维素上的非有丝分裂细胞的细胞质进行分离，发现一个主要的波形蛋白激酶活性（峰I）。从有丝分裂选择的细胞中分离胞浆产生了类似的活性（峰I）和额外的波形蛋白激酶活性（峰II），这在非有丝分裂制剂中没有发现。基于底物特异性和缺乏对特征抑制剂的抑制，半纯化峰I和II波形蛋白激酶活性似乎是与camp无关的酶，与酪蛋白激酶I和II不同。磷酸肽定位研究表明，峰I和峰II波形蛋白激酶磷酸化波形蛋白nh2末端区域的胰蛋白酶肽，这些肽在完整细胞中被磷酸化。用两种半纯化激酶磷酸化重组的波形蛋白细丝的电镜检查表明，磷酸化诱导了细丝的分解。这些实验表明，在有丝分裂过程中，波形蛋白磷酸化的增加可能是由一种独立于camp的蛋白激酶催化的。此外，有丝分裂细胞的制剂显示出Ca2+刺激的磷酸酶活性，这表明Ca2+可能在有丝分裂过程中对波形蛋白去磷酸化起调节作用。
The phosphorylation of the intermediate filament protein vimentin was examined under in vitro conditions. Cell cytosol and Triton-insoluble cytoskeleton preparations from nonmitotic and mitotically selected mouse L-929 cells exhibited vimentin kinase activity that is apparently cAMP and Ca2+ independent. The level of vimentin kinase activity was greater in preparations from mitotically selected cells than nonmitotic cells. Addition of Ca2+ to mitotic cytosol decreased net vimentin phosphorylation. Dephosphorylation experiments indicated that there is phosphatase activity in these preparations which is stimulated by addition of Ca2+. Fractionation of cytosol from nonmitotic cells on DEAE-Sephacel and phosphocellulose revealed a single major vimentin kinase activity (peak I). Fractionation of cytosol from mitotically selected cells yielded a similar activity (peak I) and an additional vimentin kinase activity (peak II) that was not found in nonmitotic preparations. Based on substrate specificity and lack of inhibition to characteristic inhibitors, the semipurified peak I and II vimentin kinase activities appear to be cAMP-independent enzymes that are distinct from casein kinases I and II. Phosphopeptide mapping studies indicated that both peak I and peak II vimentin kinases phosphorylate tryptic peptides in the NH2-terminal region of vimentin that are phosphorylated in intact cells. Electron microscopic examination of reconstituted vimentin filaments phosphorylated with both semipurified kinases indicated that phosphorylation induced filament disassembly. These experiments indicate that the increased phosphorylation of vimentin during mitosis may be catalyzed by a discrete cAMP-independent protein kinase. In addition, preparations from mitotic cells exhibited a Ca2+-stimulated phosphatase activity, suggesting that Ca2+ may play a regulatory role in vimentin dephosphorylation during mitosis.