Adaptor Protein Ruk/CIN85 is Associated with a Subset of COPI‐Coated Membranes of the Golgi Complex

Abstract

†These authors contributed equally to this work.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.

Lack of CD2AP disrupts Glut4 trafficking and attenuates glucose uptake in podocytes

“…Accordingly, live-cell imaging illustrated that the majority of endocytosed Glut4 does not traffic back to the plasma membrane, instead remaining in a perinuclear cluster in insulin-stimulated CD2AP −/− podocytes. CD2AP has been shown to colocalize in the perinuclear region with key proteins that participate in the formation of vesicles, including COPI, clathrin and Rab5 (Havrylov et al, 2008). We found that CD2AP co-fractionates in the same subcellular fractions as the GSV markers IRAP, sortilin and Glut4 itself.…”

“…CD2AP is known to colocalize with the endoplasmic reticulum (ER) marker calnexin and the Golgi marker GM130 (also known as GOLGA2) in HeLa cells (Havrylov et al, 2008). This, together with the results of live-cell imaging, led us to investigate whether CD2AP is observed in the same sub-cellular compartments as Glut4 and Glut4 storage vesicle (GSV) markers in WT podocytes.…”

“…Welsch et al (2005) showed that CD2AP is present in a specific late endosomal compartment in podocytes and that it is involved in endosomal sorting and/or trafficking by regulating the assembly of actin on vesicles. CD2AP has also been shown to colocalize with coat protein I (COPI) vesicles and clathrin (Havrylov et al, 2008). The above data suggest that CD2AP has a role for in vesicle trafficking, and the association of CD2AP with proteins involved in insulin signalling and Glut4 trafficking led us to investigate the role of CD2AP in the regulation of glucose homeostasis.…”

“…CD2AP is a ubiquitously expressed cytoplasmic protein that functions as a scaffolding protein in different signalling and vesicle trafficking pathways (Huber et al, 2003;Schiffer et al, 2004;Kobayashi et al, 2004;Havrylov et al, 2008;Wasik et al, 2012). It is necessary for the formation of slit diaphragms and glomerular ultrafiltration, as CD2AP-knockout mice die at the age of 6 weeks due to renal failure (Shih et al, 1999).…”

CIN85/RukL Is a Novel Binding Partner of Nephrin and Podocin and Mediates Slit Diaphragm Turnover in Podocytes

“…4D, right panel (16). Other studies show that CIN85/Ruk L is associated with the Golgi complex, which takes part in membrane trafficking (26,27). We overexpressed CIN85-GFP with ␤1,4-galactotransferase-pDsRed, a Golgi-associated protein, in CD2AP ϩ/ϩ podocyte and stimulated the cells for 60 min with FGF-4.…”