PROTEIN-KINASE-C REGULATES MARCKS CYCLING BETWEEN THE PLASMA-MEMBRANE AND LYSOSOMES IN FIBROBLASTS
PROTEIN-KINASE-C REGULATES MARCKS CYCLING BETWEEN THE PLASMA-MEMBRANE AND LYSOSOMES IN FIBROBLASTS
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DOI：
10.1002/j.1460-2075.1995.tb07094.x
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发表时间：
1995-03-15
期刊：
影响因子：
11.4
通讯作者：
ADEREM, A
中科院分区：
文献类型：
--
作者：
ALLEN, LAH;ADEREM, A
MARCKS是一种蛋白激酶C（PKC）底物，在神经分泌、吞噬细胞活化和生长因子依赖性有丝分裂过程中被磷酸化。MARCKS结合钙/钙调蛋白并交联F-肌动蛋白，这两种活性都受到PKC依赖性磷酸化的调节。我们在这里提出的证据表明，PKC依赖的磷酸化也调节MARCKS之间的质膜和Lamp-1阳性溶酶体的循环。免疫荧光和免疫电子显微镜，和亚细胞分级，表明MARCKS主要是与静息成纤维细胞的质膜。PKC的激活导致MARCKS磷酸化及其从质膜到Lamp-1阳性溶酶体的位移。MARCKS磷酸化是其转运至溶酶体所必需的，因为突变被PKC磷酸化的丝氨酸残基（phos（-））或PKC抑制剂星形孢菌素，阻止MARCKS磷酸化、其从质膜释放以及其随后与溶酶体的结合。在存在促溶酶体剂或诺考达唑的情况下，MARCKS在溶酶体上积累并在药物去除后返回质膜，进一步表明蛋白质在质膜和溶酶体之间循环。与野生型MARCKS相反，磷酸（-）突变体在用NH 4Cl处理的细胞中不会在溶酶体上积累，这表明MARCKS的基础磷酸化促进了其在这两个隔室之间的组成性循环。
MARCKS is a protein kinase C (PKC) substrate that is phosphorylated during neurosecretion, phagocyte activation and growth factor-dependent mitogenesis. MARCKS binds calcium/calmodulin and crosslinks F-actin, and both these activities are regulated by PKC-dependent phosphorylation. We present evidence here that PKC-dependent phosphorylation also regulates the cycling of MARCKS between the plasma membrane and Lamp-1-positive lysosomes. Immunofluorescence and immunoelectron microscopy, and subcellular fractionation, demonstrated that MARCKS was predominantly associated with the plasma membrane of resting fibroblasts. Activation of PKC resulted in MARCKS phosphorylation and its displacement from the plasma membrane to Lamp-1-positive lysosomes. MARCKS phosphorylation is required for its translocation to lysosomes since mutating either the serine residues phosphorylated by PKC (phos(-)) or the PKC inhibitor staurosporine, prevented MARCKS phosphorylation, its release from the plasma membrane, and its subsequent association with lysosomes. In the presence of lysosomotropic agents or nocodazole, MARCKS accumulated on lysosomes and returned to the plasma membrane upon drug removal, further suggesting that the protein cycles between the plasma membrane and lysosomes. In contrast to wild-type MARCKS, the phos(-) mutant did not accumulate on lysosomes in cells treated with NH4Cl, suggesting that basal phosphorylation of MARCKS promotes its constitutive cycling between these two compartments.