van Tetering Geert, van Diest Paul, Verlaan Ingrid, van der Wall Elsken, Kopan Raphael, Vooijs Marc
Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
J Biol Chem. 2009 Nov 6;284(45):31018-27. doi: 10.1074/jbc.M109.006775. Epub 2009 Sep 2.
Notch signaling is controlled by ligand binding, which unfolds a negative control region to induce proteolytic cleavage of the receptor. First, a membrane-proximal cleavage is executed by a metalloprotease, removing the extracellular domain. This allows gamma-secretase to execute a second cleavage within the Notch transmembrane domain, which releases the intracellular domain to enter the nucleus. Here we show that the ADAM10 metalloprotease Kuzbanian, but not ADAM17/tumor necrosis factor alpha-converting enzyme, plays an essential role in executing ligand-induced extracellular cleavage at site 2 (S2) in cells and localizes this step to the plasma membrane. Importantly, genetic or pharmacological inhibition of metalloproteases still allowed extracellular cleavage of Notch, indicating the presence of unknown proteases with the ability to cleave at S2. Gain of function mutations identified in human cancers and in model organisms that map to the negative control region alleviate the requirement for ligand binding for extracellular cleavage to occur. Because cancer-causing Notch1 mutations also depend on (rate-limiting) S2 proteolysis, the identity of these alternative proteases has important implications for understanding Notch activation in normal and cancer cells.
Notch信号通路受配体结合调控，配体结合会展开一个负调控区域以诱导受体的蛋白水解切割。首先，膜近端切割由金属蛋白酶执行，去除细胞外结构域。这使得γ-分泌酶能够在Notch跨膜结构域内执行第二次切割，从而释放细胞内结构域进入细胞核。在此，我们表明ADAM10金属蛋白酶库兹班氏体（而非ADAM17/肿瘤坏死因子α转换酶）在细胞中执行配体诱导的位点2（S2）细胞外切割过程中起关键作用，并将这一步骤定位于质膜。重要的是，对金属蛋白酶的基因或药理学抑制仍允许Notch进行细胞外切割，这表明存在能够在S2位点进行切割的未知蛋白酶。在人类癌症和模式生物中鉴定出的功能获得性突变定位于负调控区域，这些突变减轻了细胞外切割发生对配体结合的需求。由于致癌的Notch1突变也依赖于（限速的）S2蛋白水解，这些替代蛋白酶的身份对于理解正常细胞和癌细胞中的Notch激活具有重要意义。
