Demonstration that the leukocyte common antigen CD45 is a protein tyrosine phosphatase.
Demonstration that the leukocyte common antigen CD45 is a protein tyrosine phosphatase.
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证明白细胞共同抗原CD45是一种蛋白酪氨酸磷酸酶。
DOI：
10.1021/bi00424a001
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Walsh,KA
中科院分区：
文献类型：
--
作者：
Tonks,NK;Charbonneau,H;Diltz,CD;Fischer,EH;Walsh,KA
生物化学系，华盛顿大学，西雅图，华盛顿98195 1988年10月12日接收; 1988年10月19日接收修订的Mandarin pt摘要：基于氨基酸序列同源性，已经提出白细胞共同抗原CD 45代表催化活性的受体连接蛋白酪氨酸磷酸酶家族[Charbonneau，H.，Tonks，N. K.，沃尔什，KA，& Fischer，E. H.等人（1988）Proc. Acad. Sci. USA 85，7182-7186]。本研究证实CD 45具有内在的蛋白酪氨酸磷酸酶活性。首先，小鼠抗CD 45单克隆抗体（mAb 9.4）通过沉淀从含有CD 45的高Mr级分中特异性消除PTB活性，所述高Mr级分通过凝胶过滤（Sephacryl S200）人脾的Triton X-100提取物制备。第二，在用高pH缓冲液从由相同抗体构建的亲和柱洗脱的高度纯化的CD 45制剂中证明了PTB活性。第三，在蔗糖密度梯度离心中，仅在通过Western分析确定的含有CD 45的那些级分中发现PTB活性。当CD 45聚集时，首先通过与mAb 9.4反应，然后加入第二个交联的抗小鼠mAb，PTK活性转移到含有CD 45的相同的较高Mr组分。加入对照IgG 2a后，未观察到CD 45或PTB的变化。在此基础上，得出结论，CD 45是一种蛋白酪氨酸磷酸酶。酪氨酰残基上蛋白质的磷酸化是癌基因产物如src的肿瘤转化的必要条件（Synder等，1985）、mos（Hannink & Donoghue，1985）和fps（Weinmaster等人，1986年）。它也是胰岛素的许多作用的介导中的重要组分（Ellis等人，1986; Chou等人，1987; Morgan & Roth，1987）和生长因子如EGF（Chou等，1987; Moolenaar等人，1988年）在多种细胞类型中。显然，蛋白质的磷酸化状态反映了其激酶和磷酸酶的相对活性。因此，蛋白酪氨酸磷酸酶（PTPases）的特性可能有助于了解参与控制正常和肿瘤细胞生长的机制。最近，人胎盘中最丰富的PTPases已被纯化至表观均一性（Tonks等人，1987年，1988年a）。这些酶作为单体单位具有活性
Department of Biochemistry, University of Washington, Seattle, Washington 98195 Received October 12, 1988; Revised Manuscript Received October 19, 1988 abstract: It has been proposed on the basis of amino acid sequence homology that the leukocyte common antigen CD45 represents a family of catalytically active, receptor-linked protein tyrosine phosphatases [Charbonneau, H., Tonks, N. K., Walsh, KA, & Fischer, E. H.(1988) Proc. Natl. Acad. Sci. USA 85, 7182-7186]. Thepresent study confirmsthat CD45 possesses intrinsic protein tyrosine phosphatase (PTPase) activity. First, a mouse monoclonal antibody to CD45 (mAb 9.4) specifically eliminated, by precipitation, PTPase activity from a high Mr fraction containing CD45, prepared by gel filtration (Sephacryl S200) of a Triton X-100 extract of human spleen. Second, PTPase activity was demonstrated in a highly purified preparation of CD45 that was eluted with a high pH buffer from an affinity column, constructed from the same antibody. Third, on sucrose density gradient centrifugation, PTPase activity was only found in those fractions that contained CD45 as determined by Western analysis. When CD45 was caused to aggregate, first by reacting it withmAb 9.4 and then adding a secondary, cross-linking anti-mouse mAb, the PTPase activity shifted to the same higher Mr fractions that contained CD45. No shift in CD45 or PTPase was observed following addition of a control IgG2a. On this basis, it is concluded that CD45 is a protein tyrosine phosphatase. e phosphorylation of proteins on tyrosyl residues is a re-quirement for neoplastic transformation byoncogene products such as src (Synder et al., 1985), mos (Hannink & Donoghue, 1985), and fps (Weinmaster et al., 1986). It is also an essential component in the mediation of many of the effects of insulin (Ellis et al., 1986; Chou et al., 1987; Morgan & Roth, 1987) and growth factors such as EGF (Chou et al., 1987; Moolenaar et al., 1988) in a variety of cell types. Clearly, thestate of phosphorylation of a protein reflects the relative activities of its kinases and phosphatases. Thus the characterization of protein tyrosine phosphatases (PTPases) may facilitate an understanding of mechanisms involved in controlling both normal and neoplastic cell growth. Recently, themost abundant PTPases of human placenta have been purified to apparent homogeneity (Tonks et al., 1987, 1988a). These enzymes are active as monomeric units