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首页> 外文期刊>Molecular and Cellular Biology >Highly Purified pp60src Induces the Actin Transformation in Microinjected Cells and Phosphorylates Selected Cytoskeletal Proteins In Vitro
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Highly Purified pp60src Induces the Actin Transformation in Microinjected Cells and Phosphorylates Selected Cytoskeletal Proteins In Vitro

机译：高度纯化的pp60src诱导微注射细胞中的肌动蛋白转化，并磷酸化体外选择的细胞骨架蛋白。

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The src gene product of Rous sarcoma virus (pp60src) was highly purified from a rat tumor cell line and shown to have physiological actin transformation activity in a cellular microinjection assay that measures the dissolution of actin microfilament bundles in vivo. The purified pp60src fraction consisted of two major proteins, seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels: a 60,000-dalton (60K) protein, identified as pp60src by immunoprecipitation with tumor-bearing rabbit immunoglobulin G (IgG) and peptide mapping, and an unrelated 65K protein. There was no evidence for proteolytic cleavage of pp60src. A 7,000-fold purification of the tyrosine-specific protein kinase activity of pp60src was achieved by this procedure. Purified pp60src phosphorylated tumor-bearing rabbit IgG heavy chains, casein, histones H1 and H2B, tubulin, and microtubule-associated proteins when assayed in vitro. When incubated with [γ-32P]ATP in the absence of exogenous phosphoacceptor substrates, purified pp60src became labeled with 32P at the tyrosine residues exclusively. Phosphatase and cyclic AMP-dependent protein kinase activities were undetectable in the purified fraction. Microinjection of highly purified pp60src into the cytoplasm of normal Swiss 3T3 mouse fibroblasts caused rapid and reversible dissolution of actin stress fibers, as visualized by indirect immunofluorescence with actin antibodies. The actin-disrupting activity was thermolabile and sensitive to inhibition by coinjection of tumor-bearing rabbit IgG, and purified to about the same extent (8,000-fold) as did the IgG kinase activity of pp60src, thus implicating pp60src as the active agent. Examination of actin-associated proteins as substrates for the pp60src kinase in vitro showed that vinculin was phosphorylated directly by pp60src, although to a small extent. Actin, myosin, and tropomyosin were not phosphorylated. Thus, pp60src purified by this procedure retains native functional properties and provides a useful probe for analyzing transformation-dependent changes in actin cytoarchitecture.
机译：劳斯肉瘤病毒的 src 基因产物（pp60 src ）已从大鼠肿瘤细胞系中高度纯化，并具有生理肌动蛋白转化活性在细胞微注射分析中测量肌动蛋白微丝束在体内的溶解。纯化的pp60 src 级分由两种主要蛋白质组成，在银染的十二烷基硫酸钠-聚丙烯酰胺凝胶上观察到：60,000道尔顿（60K）蛋白质，鉴定为pp60 < sup> src 通过带有荷瘤兔免疫球蛋白G（IgG）和肽图的免疫沉淀以及无关的65K蛋白进行。没有证据表明pp60 src 会被蛋白水解。通过该程序可对pp60 src 的酪氨酸特异性蛋白激酶活性进行7,000倍纯化。体外测定时，纯化的pp60 src 磷酸化的荷瘤兔IgG重链，酪蛋白，组蛋白H1和H2B，微管蛋白和微管相关蛋白。当在没有外源磷酸受体底物的情况下与[γ- 32 P] ATP孵育时，纯化的pp60 src 被标记为 32 < / sup> P仅位于酪氨酸残基。在纯化的馏分中未检测到磷酸酶和环状AMP依赖的蛋白激酶活性。高纯度的pp60 src 的微量注射到正常的Swiss 3T3小鼠成纤维细胞的细胞质中导致肌动蛋白应激纤维的快速和可逆溶解，如通过肌动蛋白抗体的间接免疫荧光观察。破坏肌动蛋白的活性是热不稳定的，并且对带有荷瘤兔IgG的共注射抑制作用敏感，并且纯化的程度与pp60 src ，因此暗示pp60 src 作为活性剂。体外检查肌动蛋白相关蛋白作为pp60 src 激酶的底物，表明纽蛋白直接被pp60 src 磷酸化。 sup>，尽管程度很小。肌动蛋白，肌球蛋白和原肌球蛋白未磷酸化。因此，通过此程序纯化的pp60 src 保留了天然的功能特性，并为分析肌动蛋白细胞结构中依赖于转化的变化提供了有用的探针。

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