Swierczynski S L, Blackshear P J
Howard Hughes Medical Institute, Durham, North Carolina 27710, USA.
J Biol Chem. 1995 Jun 2;270(22):13436-45. doi: 10.1074/jbc.270.22.13436.
The myristoylated alanine-rich C kinase substrate (MARCKS) protein, a prominent cellular substrate for protein kinase C, is associated with membranes in various cell types. MARCKS is myristoylated at its amino terminus; this modification is thought to play the major role in anchoring MARCKS to cellular membranes. Recent studies have suggested that the protein's basic phosphorylation site/calmodulin binding domain may also be involved in the membrane association of MARCKS through electrostatic interactions. The present studies used mutations in the primary structure of the protein to investigate the nature of the association between MARCKS and cell membranes. In chick embryo fibroblasts, activation of protein kinase C led to a decrease in MARCKS membrane association as determined by cell fractionation techniques. Cell-free assays revealed that nonmyristoylated MARCKS exhibited almost no affinity for fibroblast membranes, despite readily demonstrable binding of the wild-type protein. Similar experiments in which the four serines in the phosphorylation site domain were mutated to aspartic acids, mimicking phosphorylation, decreased, but did not eliminate, membrane binding when compared to either the wild-type protein or a comparable tetra-asparagine mutant. Addition of calmodulin in the presence of Ca2+ also inhibited binding of the wild-type protein to membranes, presumably by neutralizing the phosphorylation site domain, or by physically interfering with its membrane association. Surprisingly, expression of a nonmyristoylatable mutant form of MARCKS in intact cells led to only a 46% decrease in its plasma membrane association, as determined by cell fractionation and immunoelectron microscopy. These results are consistent with a complex model of the interaction of MARCKS with cellular membranes, in which the myristoyl moiety, the positively charged phosphorylation site domain, and possibly other domains make independent contributions to membrane binding in intact cells.
富含豆蔻酰化丙氨酸的蛋白激酶C底物（MARCKS）蛋白是蛋白激酶C的一种重要细胞底物，与多种细胞类型的细胞膜相关。MARCKS在其氨基末端被豆蔻酰化；这种修饰被认为在将MARCKS锚定到细胞膜中起主要作用。最近的研究表明，该蛋白的碱性磷酸化位点/钙调蛋白结合结构域也可能通过静电相互作用参与MARCKS与细胞膜的结合。本研究利用该蛋白一级结构中的突变来研究MARCKS与细胞膜之间结合的性质。在鸡胚成纤维细胞中，通过细胞分级分离技术测定，蛋白激酶C的激活导致MARCKS与细胞膜的结合减少。无细胞实验表明，非豆蔻酰化的MARCKS对成纤维细胞膜几乎没有亲和力，尽管野生型蛋白的结合很容易被证实。在类似实验中，将磷酸化位点结构域中的四个丝氨酸突变为天冬氨酸以模拟磷酸化，与野生型蛋白或类似的四聚天冬酰胺突变体相比，膜结合减少但并未消除。在Ca2+存在的情况下添加钙调蛋白也抑制了野生型蛋白与膜的结合，推测是通过中和磷酸化位点结构域或通过物理干扰其与膜的结合。令人惊讶的是，通过细胞分级分离和免疫电子显微镜测定，在完整细胞中表达不可豆蔻酰化的MARCKS突变体形式仅导致其质膜结合减少46%。这些结果与MARCKS与细胞膜相互作用的复杂模型一致，其中豆蔻酰部分、带正电荷的磷酸化位点结构域以及可能的其他结构域在完整细胞中对膜结合做出独立贡献。
