Braun T, McIlhinney R A, Vergères G
Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056, Basel, Switzerland.
Biochimie. 2000 Aug;82(8):705-15. doi: 10.1016/s0300-9084(00)01154-8.
The myristoylated alanine-rich C kinase substrate (MARCKS) has been proposed to regulate the plasticity of the actin cytoskeleton at its site of attachment to membranes. In macrophages, MARCKS is implicated in various cellular events including motility, adhesion and phagocytosis. In this report we show that macrophage extracts contain a protease which specifically cleaves human MARCKS, expressed in a cell-free system or in E. coli, between Lys-6 and Thr-7. Cleavage of MARCKS decreases its affinity for macrophage membranes by ca. one order of magnitude, highlighting the contribution of the myristoyl moiety of MARCKS to membrane binding. Importantly, cleavage requires myristoylation of MARCKS. Furthermore, MARCKS-related protein (MRP), the second member of the MARCKS family, is not digested. Since Thr-7 is lacking in MRP this suggests that Thr-7 at the P1 position is important for the recognition of lipid-modified substrates. A different product is observed when MARCKS is incubated with a calf brain cytosolic extract. This product can be remyristoylated in the presence of myristoyl-CoA and N-myristoyl transferase, demonstrating that cycles of myristoylation/demyristoylation of MARCKS can be achieved in vitro. Although the physiological relevance of these enzymes still needs to be demonstrated, our results reveal the presence of a new class of cleaving enzymes recognizing lipid-modified protein substrates.
富含豆蔻酰化丙氨酸的蛋白激酶C底物（MARCKS）被认为在其与膜结合的位点调节肌动蛋白细胞骨架的可塑性。在巨噬细胞中，MARCKS参与多种细胞活动，包括运动、黏附和吞噬作用。在本报告中，我们发现巨噬细胞提取物中含有一种蛋白酶，该蛋白酶可特异性切割在无细胞系统或大肠杆菌中表达的人MARCKS，切割位点在赖氨酸-6和苏氨酸-7之间。MARCKS的切割使其对巨噬细胞膜的亲和力降低约一个数量级，突出了MARCKS的豆蔻酰基部分对膜结合的作用。重要的是，切割需要MARCKS进行豆蔻酰化。此外，MARCKS家族的第二个成员——MARCKS相关蛋白（MRP）未被消化。由于MRP中缺乏苏氨酸-7，这表明P1位置的苏氨酸-7对于识别脂质修饰的底物很重要。当MARCKS与小牛脑胞质提取物一起孵育时，会观察到不同的产物。该产物在存在豆蔻酰辅酶A和N-豆蔻酰转移酶的情况下可以重新进行豆蔻酰化，这表明MARCKS的豆蔻酰化/去豆蔻酰化循环可以在体外实现。尽管这些酶的生理相关性仍需证明，但我们的结果揭示了一类新的识别脂质修饰蛋白底物的切割酶的存在。
