The shedding of membrane-anchored heparin-binding epidermal-like growth factor is regulated by the Raf/mitogen-activated protein kinase cascade and by cell adhesion and spreading
The shedding of membrane-anchored heparin-binding epidermal-like growth factor is regulated by the Raf/mitogen-activated protein kinase cascade and by cell adhesion and spreading
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DOI：
10.1074/jbc.274.40.28828
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发表时间：
1999-10-01
影响因子：
4.8
通讯作者：
Klagsbrun, M
中科院分区：
文献类型：
--
作者：
Gechtman, Z;Alonso, JL;Klagsbrun, M
肝素结合表皮样生长因子（HB-EGF）是作为跨膜前体（HB-EGF（TM））合成的。向表达HB-EGF（TM）的细胞中添加佛波酯（PMA，佛波醇12-肉豆蔻酸酯13-乙酸酯）会导致可溶性HB-EGF的金属蛋白酶依赖性释放（脱落）。为了分析调节HB-EGF脱落的机制，建立了表达HB-EGF（TM）的稳定细胞系，其中胞外域和胞质尾分别用血凝素（HA）和Myc表位标记（HB-EGF（TM）HA/Myc）。HB-EGF（TM）HA/Myc裂解后，在条件培养基中出现可溶性HB-EGF（TM）HA，生物素化的细胞表面HB-EGF（TM）HA/Myc丧失，以及在细胞裂解物中出现Myc标记的胞质尾片段。通过使用这种方法，如下鉴定了几种新的HB-EGF（TM）脱落的金属蛋白酶依赖性调节剂。(i)PMA诱导的HB-EGF（TM）HA/Myc脱落可被丝裂原活化蛋白（MAP）激酶抑制剂PD 98059阻断。PMA在5 min内激活MAP激酶，而HB-EGF（TM）HA/Myc脱落直到20 min才发生，表明MAP激酶激活是PMA诱导HB-EGF裂解途径中的必要步骤。(ii)诱导型Raf-1激酶（Delta Raf-1：雌激素受体）的激活导致MAP激酶在10分钟内快速激活，HB-EGF（TM）HA/Myc在20-40分钟内脱落。（iii）血清诱导的MAP激酶激活和HB-EGF（TM）HA/Myc脱落被PD 98059抑制。（iv）PMA诱导附着细胞中的HB-EGF（TM）HA/Myc脱落，而当细胞置于悬浮液中时不发生脱落。脱落完全恢复后不久，细胞仅仅允许在纤连蛋白上传播，PMA诱导脱落的程度随着细胞传播的程度而增加。PMA诱导相同水平的MAP激酶激活的细胞是否附着或悬浮表明，虽然MAP激酶激活可能是必要的脱落，它是不够的。两者合计，这些结果表明，有两个组件的细胞调节，有助于脱落过程中，以前没有认识到，Raf-1/MAP激酶信号转导通路和细胞粘附和扩散。
Heparin-binding epidermal-like growth factor (HB-EGF) is synthesized as a transmembrane precursor (HB-EGF(TM)). The addition of phorbol ester (PMA, phorbol 12-myristate 13-acetate) to cells expressing HB-EGF(TM) results in the metalloproteinase-dependent release (shedding) of soluble HB-EGF. To analyze mechanisms that regulate HB-EGF shedding, a stable cell line was established expressing HB-EGF(TM) in which the ectodomain and the cytoplasmic tail were tagged with hemagglutinin (HA) and Myc epitopes, respectively (HB-EGF(TM)HA/Myc). HB-EGF(TM)HA/Myc cleavage was followed by the appearance of soluble HB-EGFHA in conditioned medium, the loss of biotinylated cell surface HB-EGF(TM)HA/Myc, and the appearance of a Myc-tagged cytoplasmic tail fragment in cell lysates, By using this approach, several novel metalloproteinase-dependent regulators of HB-EGF(TM) shedding were identified as follows. (i) HB-EGF(TM)HA/Myc shedding induced by PMA was blocked by the mitogen-activated protein (MAP) kinase kinase inhibitor, PD98059. PMA activated MAP kinase within 5 min, but HB-EGF(TM)HA/Myc shedding did not occur until 20 min, suggesting that MAP kinase activation was a necessary step in the pathway of PMA-induced HB-EGF, cleavage. (ii) Activation of an inducible Raf-l kinase, Delta Raf-1:estrogen receptor, resulted in a rapid MAP kinase activation within 10 min and shedding of HB-EGF(TM)HA/Myc within 20-40 min. (iii) Serum induced MAP kinase activation and HB-EGF(TM)HA/Myc shedding that were inhibited by PD98059, (iv) Whereas PMA induced HB-EGF(TM)HA/Myc shedding in attached cells, no shedding occurred when the cells were placed in suspension. Shedding was fully restored shortly after cells mere allowed to spread on fibronectin, and the extent of PMA-induced shedding increased with the extent of cell spreading. PMA induced the same level of MAP kinase activation whether the cells were attached or in suspension suggesting that although MAP kinase activation might be necessary for shedding, it was not sufficient. Taken together, these results suggest that there are two components of cell regulation that contribute to the shedding process, not previously recognized, the Raf-1/MAP kinase signal transduction pathway and cell adhesion and spreading.