Wang Xiaobiao, Mizushima Hiroto, Adachi Satoshi, Ohishi Minako, Iwamoto Ryo, Mekada Eisuke
Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan.
Cell Struct Funct. 2006;31(1):15-27. doi: 10.1247/csf.31.15.
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.
肝素结合表皮生长因子样生长因子（HB-EGF）最初被合成成为一种锚定在质膜上的跨膜前体蛋白。细胞外的表皮生长因子样结构域在胞外结构域脱落时作为一种促有丝分裂原和促细胞运动因子发挥作用，但其跨膜结构域和细胞质结构域的功能作用在很大程度上尚不清楚。我们在此证明，HB-EGF的细胞质结构域会因外部刺激而发生磷酸化，且该磷酸化位点参与了HB-EGF依赖的肿瘤发生过程。用12-O-十四酰佛波醇-13-乙酸酯（TPA）处理过表达人HB-EGF的Vero细胞，会导致HB-EGF的胞外结构域脱落，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，HB-EGF细胞质结构域中的Ser207在TPA刺激下会发生磷酸化，产生两个具有不同磷酸化状态的C末端片段。用溶血磷脂酸、茴香霉素和钙离子载体处理细胞，这些物质均已知可诱导胞外结构域脱落，也会导致HB-EGF的磷酸化。尽管HB-EGF的胞外结构域脱落和磷酸化是协同发生的，但将Ser207替换为丙氨酸对TPA诱导的或组成型的胞外结构域脱落没有影响。将过表达HB-EGF的细胞注射到裸鼠体内表明，将Ser207替换为丙氨酸会降低HB-EGF的致瘤活性，尽管HB-EGF的细胞表面水平和胞外结构域脱落不受该突变的影响。此外，我们发现另一种表皮生长因子受体（EGFR）配体转化生长因子-α的细胞质结构域在TPA刺激下也会发生磷酸化。因此，目前的结果表明，HB-EGF和其他表皮生长因子家族生长因子的细胞质结构域具有一种由磷酸化调节的新作用。
