Havrylov Serhiy, Ichioka Fumitaka, Powell Kate, Borthwick Emma B, Baranska Jolanta, Maki Masatoshi, Buchman Vladimir L
School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3US, Wales, UK.
Traffic. 2008 May;9(5):798-812. doi: 10.1111/j.1600-0854.2008.00724.x. Epub 2008 Feb 11.
Regulator of ubiquitous kinase/Cbl-interacting protein of 85 kDa (Ruk/CIN85) and CD2-associated protein/Cas ligand with multiple SH3 domains (CD2AP/CMS) comprise a family of vertebrate adaptor proteins involved in several important cellular processes, including downregulation of activated receptor tyrosine kinases, regulation of cytoskeletal rearrangements, phosphatidylinositol 3-kinase (PI 3-kinase) signalling and apoptosis. The role of Ruk/CIN85 as a scaffold protein involved in membrane trafficking processes has been demonstrated in model cell systems. However, intracellular localization of endogenous Ruk/CIN85 has never been comprehensively assessed. We carried out detailed studies of subcellular distribution of Ruk/CIN85 in adherent cultured human cells using antibodies that recognize distinct epitopes of the protein and revealed a punctate immunostaining pattern, common for proteins involved in intracellular trafficking processes. Our data indicate that Ruk/CIN85 is distributed between several different membrane trafficking compartments, but the major pool of Ruk/CIN85 is associated with the Golgi complex, mainly with a subpopulation of COPI-coated vesicles involved in retrograde endoplasmic reticulum-Golgi and intra-Golgi transport. This localization pattern is dependent on the integrity of Golgi complex and intact microtubular network. Only a small pool of Ruk/CIN85 is present in compartments involved in clathrin-mediated endocytosis and sorting. These results suggest that endogenous Ruk/CIN85 may be involved in regulation of specific membrane trafficking processes.
泛在激酶调节因子/85 kDa的Cbl相互作用蛋白（Ruk/CIN85）和具有多个SH3结构域的CD2相关蛋白/Cas配体（CD2AP/CMS）构成了一个脊椎动物衔接蛋白家族，参与多种重要的细胞过程，包括活化受体酪氨酸激酶的下调、细胞骨架重排的调节、磷脂酰肌醇3激酶（PI 3激酶）信号传导和细胞凋亡。在模型细胞系统中已证明Ruk/CIN85作为参与膜运输过程的支架蛋白的作用。然而，内源性Ruk/CIN85的细胞内定位从未得到全面评估。我们使用识别该蛋白不同表位的抗体，对贴壁培养的人细胞中Ruk/CIN85的亚细胞分布进行了详细研究，发现了点状免疫染色模式，这是参与细胞内运输过程的蛋白所共有的。我们的数据表明，Ruk/CIN85分布在几个不同的膜运输区室之间，但Ruk/CIN85的主要部分与高尔基体复合体相关，主要与参与内质网-高尔基体逆行运输和高尔基体内运输的COPI包被小泡亚群相关。这种定位模式依赖于高尔基体复合体的完整性和完整的微管网络。只有一小部分Ruk/CIN85存在于参与网格蛋白介导的内吞作用和分选的区室中。这些结果表明内源性Ruk/CIN85可能参与特定膜运输过程的调节。
