CD2AP Regulates SUMOylation of CIN85 in Podocytes

Abstract

dPodocytes are highly differentiated and polarized epithelial cells located on the visceral side of the glomerulus. They form an indispensable component of the glomerular filter, the slit diaphragm, formed by several transmembrane proteins and adaptor molecules. Disruption of the slit diaphragm can lead to massive proteinuria and nephrotic syndrome in mice and humans. CD2AP is an adaptor protein that is important for the maintenance of the slit diaphragm. Together with its paralogue, CIN85, CD2AP belongs to a family of adaptor proteins that are primarily described as being involved in endocytosis and downregulation of receptor tyrosine kinase activity. We have shown that full-length CIN85 is upregulated in podocytes in the absence of CD2AP, whereas in wild-type cells, full-length CIN85 is not detectable. In this study, we show that full-length CIN85 is postranslationally modified by SUMOylation in wild-type podocytes. We can demonstrate that CIN85 is SUMOylated by SUMO-1, -2, and -3 and that SUMOylation is enhanced in the presence of CD2AP. Conversion of lysine 598 to arginine completely abolishes SUMOylation and leads to increased binding of CIN85 to nephrin. Our results indicate a novel role for CD2AP in regulating posttranslational modification of CIN85.T he adaptor molecules CD2-associated protein (CD2AP) and Cbl-interacting protein of 85 kDa (CIN85) belong to a ubiquitously expressed protein family of adaptor molecules that are involved in a variety of cellular processes, like cell signaling (12, 18, 52), cytoskeletal arrangement (2, 16, 29, 50), and degradative trafficking and endocytosis of receptors (15,24,26,43,45,49,57). The two proteins show high sequence and structural similarities, and they both contain three SH3 domains, a proline-rich region, and a coiled-coil domain (7). However, they appear to have completely different functional roles. While CD2AP is solely expressed in its full-length form, multiple CIN85/Ruk isoforms were identified in various tissues and cell lines, due to alternative splicing and different promoters (3,31).In podocytes CD2AP is expressed at the slit diaphragm, a specialized intercellular junction between neighboring podocytes covering the outer surface of the glomerular tuft. CD2AP interacts with several proteins at the slit diaphragm. One of the major components is nephrin, a transmembrane adhesion protein of the Ig superfamily. Humans and mice lacking nephrin are born without intact slit diaphragms and develop massive proteinuria in utero (22,40). Mice deficient in CD2AP are born healthy but develop a rapid-onset nephrotic syndrome at 3 weeks of age and die of renal failure 6 weeks after birth (44). We have previously demonstrated that deficiency of CD2AP leads to a differentiation-dependent increase of full-length CIN85 expression, which correlates with a loss of expression of the slit diaphragm protein nephrin in podocytes. Furthermore, we found that CIN85 is a binding partner of nephrin and that overexpression of CIN85 leads to increased endocytosis of nephrin after gr...

Spatiotemporal distribution of SUMOylation components during mouse brain development

“…Our immunofluorescent staining of cultured mouse neural stem cells (see Fig. ) produced a pattern of SUMO1 and SUMO2/3 immunoreactivity identical to previous descriptions (Hashiguchi et al, ; Tossidou et al, ).…”

An update: the role of Nephrin inside and outside the kidney

“…The N-terminal domain of CD2AP can bind to p85 and facilitate the Nephrin-induced AKT signaling [22], which protects podocytes from apoptosis. CD2AP enhanced the small ubiquitin-related modification of CIN85, which increase the binding of CIN85 to Nephrin [23], induces Nephrin ubiquitination and endocytosis [24].CD2AP knockout (CD2AP  ) mice are born healthy but develop a rapid-onset nephrotic syndrome at three weeks of age and die of renal failure at six weeks [21,25]. Kidneys from CD2AP  mice initially exhibit normal Nephrin localization, but with aging the FP become effaced [26] and deficiency of CD2AP leads to a loss of expression of the SD protein Nephrin in podocytes.…”

The function of endocytosis in podocytes

“…However, in podocytes, CD2AP and CIN85 have functionally compensatory roles. In wild type podocytes, CD2AP appears to downregulate CIN85 expression through posttranslational modification by SUMOylation at lysine 598 (65). Conversely in podocytes lacking CD2AP expression, there is increased CIN85/RukL expression, which results in ubiquitination of nephrin.…”