Cytoplasmic domain phosphorylation of heparin-binding EGF-like growth factor
Cytoplasmic domain phosphorylation of heparin-binding EGF-like growth factor
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DOI：
10.1247/csf.31.15
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发表时间：
2006-01-01
影响因子：
1.5
通讯作者：
Mekada, Eisuke
中科院分区：
文献类型：
--
作者：
Wang, Xiaobiao;Mizushima, Hiroto;Mekada, Eisuke
肝素结合EGF样生长因子（HB-EGF）是作为锚定在质膜上的跨膜前体蛋白合成的。细胞外EGF样结构域作为有丝分裂原和运动后，胞外域脱落，但跨膜和胞质结构域的功能作用在很大程度上是未知的。我们在这里表明，HB-EGF的胞质结构域被磷酸化的外部刺激，磷酸化位点参与HB-EGF依赖性肿瘤的发生。用12-O-十四酰基佛波醇-13-乙酸酯（TPA）处理过表达人HB-EGF的Vero细胞，引起HB-EGF的胞外域脱落，并产生两个具有不同电泳迁移率的羧基（C）末端片段。突变分析表明，Ser 207在HB-EGF的胞质结构域磷酸化TPA刺激后，产生两个C-末端片段具有不同的磷酸化状态。溶血磷脂酸，茴香霉素，钙离子载体，所有这些都是已知的诱导胞外域脱落的细胞治疗，也引起HB-EGF的磷酸化。虽然胞外域脱落和HB-EGF的磷酸化协同发生，但Ala取代Ser 207对TPA诱导的或组成性胞外域脱落没有影响。将过表达HB-EGF的细胞注射到裸鼠体内，结果表明，Ala取代Ser 207降低了HB-EGF的致瘤活性，即使HB-EGF的细胞表面水平和胞外域脱落不受突变的影响。此外，我们发现另一种EGFR配体转化生长因子-α的胞质结构域在TPA刺激后被磷酸化。因此，目前的研究结果表明，HB-EGF和其他EGF家族生长因子的细胞质结构域的一个新的作用是由磷酸化调节。
Heparin-binding EGF-like growth factor (HB-EGF) is synthesized as a transmembrane precursor protein that is anchored to the plasma membrane. The extracellular EGF-like domain acts as a mitogen and motogen upon ectodomain shedding, but the functional roles of the transmembrane and cytoplasmic domains are largely unknown. We demonstrate here that cytoplasmic domain of HB-EGF is phosphorylated by external stimuli, and that the phosphorylation site is involved in HB-EGF-dependent tumorigenesis. Treatment of Vero cells overexpressing human HB-EGF with 12-O-tetradecanoylphorbol-13-acetate (TPA) caused ectodomain shedding of HB-EGF and generated two carboxyl (C)-terminal fragments with distinct electrophoretic mobilities. Mutation analysis showed that Ser207 in the cytoplasmic domain of HB-EGF is phosphorylated upon TPA stimulation, generating two C-terminal fragments with distinct phosphorylation states. Treatment of cells with lysophosphatidic acid, anisomycin, and calcium ionophore, all of which are known to induce ectodomain shedding, also caused phosphorylation of HB-EGF. Although ectodomain shedding and phosphorylation of HB-EGF occurred coordinately, Ala substitution of Ser207 had no effect on TPA-induced or constitutive ectodomain shedding. Injection of cells overexpressing HB-EGF into nude mice showed that Ala substitution of Ser207 reduced the tumorigenic activity of HB-EGF, even though the cell surface level and ectodomain shedding of HB-EGF were not affected by the mutation. Moreover, we found that the cytoplasmic domain of another EGFR ligand, transforming growth factor-alpha, is phosphorylated upon TPA stimulation. Thus, the present results suggest a novel role for the cytoplasmic domain of HB-EGF and other EGF family growth factors that is regulated by phosphorylation.