Spizz G, Blackshear P J
Howard Hughes Medical Institute, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 1997 Sep 19;272(38):23833-42. doi: 10.1074/jbc.272.38.23833.
The importance of regulating the cellular concentrations of the myristoylated alanine-rich C kinase substrate (MARCKS), a major cellular substrate of protein kinase C, is indicated by the fact that mice lacking MARCKS exhibit gross abnormalities of central nervous system development and die shortly after birth. We previously identified a novel means of regulating cellular MARCKS concentrations that involved a specific proteolytic cleavage of the protein and implicated a cysteine protease in this process (Spizz, G., and Blackshear, P. J. (1996) J. Biol. Chem. 271, 553-562). Here we show that p40, the carboxyl-terminal fragment resulting from this cleavage of MARCKS, was associated with the mitochondrial/lysosomal pellet fraction of human diploid fibroblasts and that its generation in cells was sensitive to treatment with NH4Cl. These data suggest the involvement of lysosomes in the generation and/or stability of p40. The MARCKS-cleaving enzyme (MCE) activity was peripherally associated with a 10,000 x g pellet fraction from bovine liver, and it co-purified with the activity and immunoreactivity of a lysosomal protease, cathepsin B. Cathepsin B catalyzed the generation of p40 from MARCKS in a cell-free system and behaved similarly to the MCE with respect to mutants of MARCKS previously shown to be poor substrates for the MCE. Treatment of fibroblasts with a cell-permeable, specific inhibitor of cathepsin B, CA074-Me, resulted in parallel time- and concentration-dependent inhibition of cathepsin B and MCE activity. Incubation of a synthetic MARCKS phosphorylation site domain peptide with purified cathepsin B resulted in cleavage of the peptide at sites consistent with preferred cathepsin B substrate sites. These data provide evidence for the identity of the MCE as cathepsin B and suggest that this cleavage most likely takes place within lysosomes, perhaps as a result of specific lysosomal targeting sequences within the MARCKS primary sequence. The data also suggest a direct interaction between MARCKS and cathepsin B in cells and leave open the possibility that MARCKS may in some way regulate the protease for which it is a substrate.
蛋白激酶C的主要细胞底物——富含肉豆蔻酰化丙氨酸的蛋白激酶C底物（MARCKS）的细胞浓度调节至关重要，这一事实表明，缺乏MARCKS的小鼠表现出中枢神经系统发育的严重异常，并在出生后不久死亡。我们之前发现了一种调节细胞MARCKS浓度的新方法，该方法涉及该蛋白的特异性蛋白水解切割，并在此过程中涉及一种半胱氨酸蛋白酶（Spizz, G., and Blackshear, P. J. (1996) J. Biol. Chem. 271, 553 - 562）。在此我们表明，p40是MARCKS这种切割产生的羧基末端片段，它与人二倍体成纤维细胞的线粒体/溶酶体沉淀部分相关，并且其在细胞中的产生对用NH4Cl处理敏感。这些数据表明溶酶体参与了p40的产生和/或稳定性。MARCKS切割酶（MCE）活性与来自牛肝的10,000×g沉淀部分外周相关，并且它与溶酶体蛋白酶组织蛋白酶B的活性和免疫反应性共纯化。组织蛋白酶B在无细胞系统中催化从MARCKS产生p40，并且对于先前显示为MCE不良底物的MARCKS突变体，其行为与MCE相似。用细胞可渗透的组织蛋白酶B特异性抑制剂CA074-Me处理成纤维细胞，导致组织蛋白酶B和MCE活性平行的时间和浓度依赖性抑制。将合成的MARCKS磷酸化位点结构域肽与纯化的组织蛋白酶B一起孵育，导致该肽在与组织蛋白酶B优选底物位点一致的位点处切割。这些数据为MCE与组织蛋白酶B的同一性提供了证据，并表明这种切割很可能发生在溶酶体内，可能是由于MARCKS一级序列中的特定溶酶体靶向序列。数据还表明细胞中MARCKS与组织蛋白酶B之间存在直接相互作用，并留下了MARCKS可能以某种方式调节其作为底物的蛋白酶的可能性。
