Mori Seiji, Tanaka Motonari, Nanba Daisuke, Nishiwaki Eiji, Ishiguro Hiroshi, Higashiyama Shigeki, Matsuura Nariaki
Department of Molecular Pathology, School of Allied Health Science, Osaka University Faculty of Medicine, 1-7 Yamadaoka, Suita, Osaka 565-0871, USA.
J Biol Chem. 2003 Nov 14;278(46):46029-34. doi: 10.1074/jbc.M306393200. Epub 2003 Sep 2.
A disintegrin and metalloprotease 12 (ADAM12/meltrin alpha) is a key enzyme implicated in the ectodomain shedding of membrane-anchored heparin-binding epidermal growth factor (EGF)-like growth factor (proHB-EGF)-dependent epidermal growth factor receptor (EGFR) transactivation. However, the activation mechanisms of ADAM12 are obscure. To determine how ADAM12 is activated, we screened proteins that bind to the cytoplasmic domain of ADAM12 using a yeast two-hybrid system and identified a protein called PACSIN3 that contains a Src homology 3 domain. An analysis of interactions between ADAM12 and PACSIN3 using glutathione S-transferase fusion protein revealed that a proline-rich region (amino acid residues 829-840) of ADAM12 was required to bind PACSIN3. Furthermore, co-immunoprecipitation and co-localization analyses of ADAM12 and PACSIN3 proteins also revealed their interaction in mammalian cells expressing both of them. The overexpression of PACSIN3 in HT1080 cells enhanced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced proHB-EGF shedding. Furthermore, knockdown of endogenous PACSIN3 by small interfering RNA in HT1080 cells significantly attenuated the shedding of proHB-EGF induced by TPA and angiotensin II. Our data indicate that PACSIN3 has a novel function as an up-regulator in the signaling of proHB-EGF shedding induced by TPA and angiotensin II.
解整合素金属蛋白酶12（ADAM12/黑素转铁蛋白α）是一种关键酶，参与膜锚定的肝素结合表皮生长因子（EGF）样生长因子（proHB-EGF）依赖性表皮生长因子受体（EGFR）反式激活的胞外域脱落过程。然而，ADAM12的激活机制尚不清楚。为了确定ADAM12是如何被激活的，我们使用酵母双杂交系统筛选了与ADAM12胞质结构域结合的蛋白质，并鉴定出一种名为PACSIN3的蛋白质，它含有一个Src同源3结构域。利用谷胱甘肽S-转移酶融合蛋白对ADAM12和PACSIN3之间的相互作用进行分析，结果表明ADAM12的富含脯氨酸区域（氨基酸残基829-840）是结合PACSIN3所必需的。此外，对ADAM12和PACSIN3蛋白进行的共免疫沉淀和共定位分析也揭示了它们在同时表达这两种蛋白的哺乳动物细胞中的相互作用。PACSIN3在HT1080细胞中的过表达增强了12-O-十四烷酰佛波醇-13-乙酸酯（TPA）诱导的proHB-EGF脱落。此外，在HT1080细胞中用小干扰RNA敲低内源性PACSIN3可显著减弱TPA和血管紧张素II诱导的proHB-EGF脱落。我们的数据表明，PACSIN3作为TPA和血管紧张素II诱导的proHB-EGF脱落信号传导中的上调因子具有新功能。
