Maness P F, Levy B T
Mol Cell Biol. 1983 Jan;3(1):102-12. doi: 10.1128/mcb.3.1.102-112.1983.
The src gene product of Rous sarcoma virus (pp60(src)) 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 pp60(src) fraction consisted of two major proteins, seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels: a 60,000-dalton (60K) protein, identified as pp60(src) 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 pp60(src). A 7,000-fold purification of the tyrosine-specific protein kinase activity of pp60(src) was achieved by this procedure. Purified pp60(src) phosphorylated tumor-bearing rabbit IgG heavy chains, casein, histones H1 and H2B, tubulin, and microtubule-associated proteins when assayed in vitro. When incubated with [gamma-(32)P]ATP in the absence of exogenous phosphoacceptor substrates, purified pp60(src) became labeled with (32)P at the tyrosine residues exclusively. Phosphatase and cyclic AMP-dependent protein kinase activities were undetectable in the purified fraction. Microinjection of highly purified pp60(src) 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 pp60(src), thus implicating pp60(src) as the active agent. Examination of actin-associated proteins as substrates for the pp60(src) kinase in vitro showed that vinculin was phosphorylated directly by pp60(src), although to a small extent. Actin, myosin, and tropomyosin were not phosphorylated. Thus, pp60(src) purified by this procedure retains native functional properties and provides a useful probe for analyzing transformation-dependent changes in actin cytoarchitecture.
从大鼠肿瘤细胞系中高度纯化了劳氏肉瘤病毒的src基因产物（pp60(src)），并在一种细胞显微注射试验中显示其具有生理肌动蛋白转化活性，该试验可在体内测量肌动蛋白微丝束的溶解情况。纯化的pp60(src)组分在银染的十二烷基硫酸钠 - 聚丙烯酰胺凝胶上可见两种主要蛋白质：一种60,000道尔顿（60K）的蛋白质，通过用荷瘤兔免疫球蛋白G（IgG）进行免疫沉淀和肽图谱分析鉴定为pp60(src)，以及一种不相关的65K蛋白质。没有证据表明pp60(src)发生了蛋白水解切割。通过该程序实现了pp60(src)酪氨酸特异性蛋白激酶活性7000倍的纯化。体外测定时，纯化的pp60(src)使荷瘤兔IgG重链、酪蛋白、组蛋白H1和H2B、微管蛋白以及微管相关蛋白发生磷酸化。当在没有外源磷酸受体底物的情况下与[γ-(32)P]ATP一起孵育时，纯化的pp60(src)仅在酪氨酸残基处被(32)P标记。在纯化组分中未检测到磷酸酶和环磷酸腺苷依赖性蛋白激酶活性。将高度纯化的pp60(src)显微注射到正常瑞士3T3小鼠成纤维细胞的细胞质中，通过用肌动蛋白抗体进行间接免疫荧光观察，导致肌动蛋白应力纤维快速且可逆地溶解。肌动蛋白破坏活性对热不稳定，并且对共注射荷瘤兔IgG的抑制敏感，并且纯化程度与pp60(src)的IgG激酶活性大致相同（8000倍），因此表明pp60(src)是活性剂。体外检查作为pp60(src)激酶底物的肌动蛋白相关蛋白表明，纽蛋白虽然程度较小，但可被pp60(src)直接磷酸化。肌动蛋白、肌球蛋白和原肌球蛋白未被磷酸化。因此，通过该程序纯化的pp60(src)保留了天然功能特性，并为分析肌动蛋白细胞结构中与转化相关的变化提供了有用的探针。
