IDENTIFICATION AND SEQUENCE-ANALYSIS OF CDNAS ENCODING A 110-KILODALTON ACTIN FILAMENT-ASSOCIATED PP60(SRC) SUBSTRATE
IDENTIFICATION AND SEQUENCE-ANALYSIS OF CDNAS ENCODING A 110-KILODALTON ACTIN FILAMENT-ASSOCIATED PP60(SRC) SUBSTRATE
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DOI：
10.1128/mcb.13.12.7892
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发表时间：
1993-12-01
影响因子：
5.3
通讯作者：
PARSONS, JT
中科院分区：
文献类型：
--
作者：
FLYNN, DC;LEU, TH;PARSONS, JT
通过pp60src的致癌形式（例如pp60v-src或pp60527F）转化鸡胚胎细胞与酪氨酸磷酸化细胞蛋白稳态水平的增加相关。Src蛋白酪氨酸激酶的活化形式稳定地与酪氨酸磷酸化蛋白结合，包括110 kDa的蛋白pp110。先前的报道已经确定pp110和pp60src之间稳定的复合物形成需要Src SH2和SH3结构域的结构完整性，而pp110的酪氨酸磷酸化只需要SH3结构域的结构完整性。在正常的鸡胚细胞中，pp110与肌动蛋白应激丝共定位，而在src转化的细胞中，pp110与足小体（莲座）相关。在这里，我们报道了编码pp110的cdna的鉴定和表征。预测的开放阅读框编码635个氨基酸的多肽，与现有序列数据库中存在的其他蛋白质序列几乎没有序列相似性。因此，pp110是一种独特的细胞骨架相关蛋白。基于其与肌动蛋白应激丝的关联，我们提出了110 kDa的肌动蛋白丝相关蛋白AFAP-110的名称。体外分析AFAP-110与细菌编码的谷胱甘肽s -转移酶（GST）融合蛋白的结合表明，正常细胞提取物中存在的AFAP-110与Src SH3/ sh2融合蛋白的结合效率高，与Src SH3/ sh2融合蛋白的结合效率较低，与sh2融合蛋白的结合效率较低。相反，在src转化的细胞提取物中，AFAP-110与GST-SH3/SH2和GST-SH2融合蛋白结合。对AFAP-110 cDNA序列的分析显示，存在预测分别结合SH2和SH3结构域的序列基序。我们认为AFAP-110可能代表一种能够与含sh3蛋白相互作用的细胞蛋白，并且在酪氨酸磷酸化后，与含sh2蛋白紧密结合，如pp60src或pp59fyn。讨论了AFAP-110作为SH3/SH2细胞骨架结合蛋白的潜在作用。
Transformation of chicken embryo cells by oncogenic forms of pp60src (e.g., pp60v-src or pp60527F) is linked with a concomitant increase in the steady-state levels of tyrosine-phosphorylated cellular proteins. Activated forms of the Src protein-tyrosine kinase stably associate with tyrosine-phosphorylated proteins, including a protein of 110 kDa, pp110. Previous reports have established that stable complex formation between pp110 and pp60src requires the structural integrity of the Src SH2 and SH3 domains, whereas tyrosine phosphorylation of pp110 requires only the structural integrity of the SH3 domain. In normal chicken embryo cells, pp110 colocalizes with actin stress filaments, and in Src-transformed cells, pp110 is found associated with podosomes (rosettes). Here, we report the identification and characterization of cDNAs encoding pp110. The predicted open reading frame encodes a polypeptide of 635 amino acids which exhibits little sequence similarity with other protein sequences present in the available sequence data bases. Thus, pp110 is a distinctive cytoskeleton-associated protein. On the basis of its association with actin stress filaments, we propose the term AFAP-110, for actin filament-associated protein of 110 kDa. In vitro analysis of AFAP-110 binding to bacterium-encoded glutathione S-transferase (GST) fusion proteins revealed that AFAP-110 present in normal cell extracts binds efficiently to Src SH3/SH2-containing fusion proteins, less efficiently to Src SH3-containing proteins, and poorly to SH2-containing fusion proteins. In contrast, AFAP-110 in Src-transformed cell extracts bound to GST-SH3/SH2 and GST-SH2 fusion proteins. Analysis of AFAP-110 cDNA sequences revealed the presence of sequence motifs predicted to bind to SH2 and SH3 domains, respectively. We suggest that AFAP-110 may represent a cellular protein capable of interacting with SH3-containing proteins and, upon tyrosine phosphorylation, binds tightly to SH2-containing proteins, such as pp60src or pp59fyn. The potential roles of AFAP-110 as an SH3/SH2 cytoskeletal binding protein are discussed.