Protein phosphatase type-1, not type-2A, modulates actin microfilament integrity and myosin light chain phosphorylation in living nonmuscle cells.
Protein phosphatase type-1, not type-2A, modulates actin microfilament integrity and myosin light chain phosphorylation in living nonmuscle cells.
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蛋白质磷酸酶类型1，而不是2A型，调节活肌动蛋白的微丝完整性和肌球蛋白轻链在活的非肌细胞中。
DOI：
10.1083/jcb.111.1.103
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发表时间：
1990-07
影响因子：
7.8
通讯作者：
Lamb, N J
中科院分区：
文献类型：
--
作者：
Fernandez, A;Brautigan, D L;Mumby, M;Lamb, N J
肌动蛋白微丝网络的动态重组依赖于肌凝蛋白轻链的可逆磷酸化。为了评估蛋白磷酸酶在活的非肌肉细胞中这一过程中的潜在作用，我们将纯化的1型和2a型磷酸酶微注射到哺乳动物成纤维细胞的细胞质中。我们的研究表明，通过免疫荧光分析，1型磷酸酶水平升高导致肌动蛋白微丝网络快速（在30分钟内）和完全可逆的分解。相比之下，微量注射等量纯化的2a型磷酸酶对肌动蛋白微丝组织没有影响。用纯化的磷酸酶注射后细胞的代谢标记来分析蛋白磷酸化的变化。在1型磷酸酶诱导肌动蛋白微丝断裂的同时，肌球蛋白轻链也发生了广泛的去磷酸化。当细胞注射2A型磷酸酶时，没有观察到这种变化。此外，用Triton X-100提取成纤维细胞后，1型磷酸酶可以通过免疫荧光特异性定位到微丝的纤维网络上。此外，通过微量注射亲和纯化抗体在体内中和1型磷酸酶活性，阻止了肌动蛋白微丝的重组，我们之前描述了在注射camp依赖性蛋白激酶后的重组。这些数据支持了1型和2型磷酸酶在活细胞中具有不同底物特异性的观点，并且1型磷酸酶在肌球蛋白轻链的去磷酸化中起主要作用，从而在完整的非肌肉细胞中调节肌动蛋白微丝组织。
Dynamic reorganization of the actin microfilament networks is dependent on the reversible phosphorylation of myosin light chain. To assess the potential role of protein phosphatases in this process in living nonmuscle cells, we have microinjected the purified type-1 and type-2A phosphatases into the cytoplasm of mammalian fibroblasts. Our studies reveal that elevating type-1 phosphatase levels led to the rapid (within 30 min) and fully reversible disassembly of the actin microfilament network as determined by immunofluorescence analysis. In contrast, microinjection of equivalent amounts of the purified type-2A phosphatase had no effect on actin microfilament organization. Metabolic labeling of cells after injection of purified phosphatases was used to analyze changes in protein phosphorylation. Concomitant with the disassembly of the actin microfilaments induced by type-1 phosphatase, there was an extensive dephosphorylation of myosin light chain. No such change was observed when cells were injected with type- 2A phosphatase. In addition, after extraction of fibroblasts with Triton X-100, the type-1 phosphatase could be specifically localized by immunofluorescence to a fibrillar network of microfilaments. Furthermore, neutralizing type-1 phosphatase activity in vivo by microinjection of an affinity-purified antibody, prevented the reorganization of actin microfilaments that we had previously described following injection of cAMP-dependent protein kinase. These data support the notion that type 1 and type-2 phosphatases have distinct substrate specificity in living cells, and that type-1 phosphatase plays a predominant role in the dephosphorylation of myosin light chain and thus in the modulation of actin microfilament organization in vivo in intact nonmuscle cells.