Proteolysis-induced N-terminal Ectodomain Shedding of the Integral Membrane Glycoprotein CUB Domain-containing Protein 1 (CDCP1) Is Accompanied by Tyrosine Phosphorylation of Its C-terminal Domain and Recruitment of Src and PKCδ
Proteolysis-induced N-terminal Ectodomain Shedding of the Integral Membrane Glycoprotein CUB Domain-containing Protein 1 (CDCP1) Is Accompanied by Tyrosine Phosphorylation of Its C-terminal Domain and Recruitment of Src and PKCδ
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DOI：
10.1074/jbc.m109.096453
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发表时间：
2010-08-20
影响因子：
4.8
通讯作者：
Hooper, John D.
中科院分区：
文献类型：
--
作者：
He, Yaowu;Wortmann, Andreas;Hooper, John D.
含CUB结构域蛋白1（CDCP 1）是一种完整的膜糖蛋白，具有作为许多癌症的标志物和治疗靶点的潜力。在这里，我们研究调节CDCP 1的细胞加工的机制。通过分析细胞系专门通过非酶促和通过使用一个面板的蛋白酶抑制剂，我们证明，全长135 kDa的CDCP 1是post-acetylated处理的一系列细胞系的机制，涉及丝氨酸蛋白酶活性，产生一个C-末端70 kDa的片段。该细胞保留片段的免疫纯化和N-末端测序以及详细的诱变显示，CDCP 1的蛋白水解加工发生在两个位点，Arg-368和Lys-369。我们表明，丝氨酸蛋白酶matriptase是一种有效的，但不是必需的，在Arg-368的CDCP 1的细胞处理器。重要的是，我们还表明，蛋白水解诱导酪氨酸磷酸化的70 kDa的CDCP 1和招聘的Src和PKC δ到这个片段。此外，蛋白质印迹和质谱分析表明，N-末端65-kDa的CDCP 1胞外域是从细胞表面脱落完整。这些数据提供了新的见解调节CDCP 1的机制，并表明这种蛋白质的生物学作用，并可能，其在癌症中的功能，可能是由70 kDa的细胞保留和65 kDa的脱落片段，以及全长135 kDa的蛋白质介导的。
CUB-domain-containing protein 1 (CDCP1) is an integral membrane glycoprotein with potential as a marker and therapeutic target for a number of cancers. Here we examine mechanisms regulating cellular processing of CDCP1. By analyzing cell lines exclusively passaged non-enzymatically and through use of a panel of protease inhibitors, we demonstrate that full-length 135 kDa CDCP1 is post-translationally processed in a range of cell lines by a mechanism involving serine protease activity, generating a C-terminal 70-kDa fragment. Immunopurification and N-terminal sequencing of this cell-retained fragment and detailed mutagenesis, show that proteolytic processing of CDCP1 occurs at two sites, Arg-368 and Lys-369. We show that the serine protease matriptase is an efficient, but not essential, cellular processor of CDCP1 at Arg-368. Importantly, we also demonstrate that proteolysis induces tyrosine phosphorylation of 70-kDa CDCP1 and recruitment of Src and PKC delta to this fragment. In addition, Western blot and mass spectroscopy analyses show that an N-terminal 65-kDa CDCP1 ectodomain is shed intact from the cell surface. These data provide new insights into mechanisms regulating CDCP1 and suggest that the biological role of this protein and, potentially, its function in cancer, may be mediated by both 70-kDa cell retained and 65-kDa shed fragments, as well as the full-length 135-kDa protein.