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CD2AP Regulates Sumoylation of CIN85 in Podocytes

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CD2AP Regulates Sumoylation of CIN85 in Podocytes Dartmouth College Dartmouth Digital Commons Dartmouth Scholarship Faculty Work 12-27-2011 CD2AP Regulates SUMOylation of CIN85 in Podocytes Irini Tossidou Harvard University Rainer Niedenthal Hannover Medical School Malte Klaus Hannover Medical School Beina Teng Harvard University Kirstin Worthmann Harvard University See next page for additional authors Follow this and additional works at: https://digitalcommons.dartmouth.edu/facoa Part of the Medical Cell Biology Commons, and the Medical Molecular Biology Commons Dartmouth Digital Commons Citation Tossidou, Irini; Niedenthal, Rainer; Klaus, Malte; Teng, Beina; Worthmann, Kirstin; King, Benjamin; and Peterson, Kevin, "CD2AP Regulates SUMOylation of CIN85 in Podocytes" (2011). Dartmouth Scholarship. 1226. https://digitalcommons.dartmouth.edu/facoa/1226 This Article is brought to you for free and open access by the Faculty Work at Dartmouth Digital Commons. It has been accepted for inclusion in Dartmouth Scholarship by an authorized administrator of Dartmouth Digital Commons. For more information, please contact [email protected]. Authors Irini Tossidou, Rainer Niedenthal, Malte Klaus, Beina Teng, Kirstin Worthmann, Benjamin King, and Kevin Peterson This article is available at Dartmouth Digital Commons: https://digitalcommons.dartmouth.edu/facoa/1226 CD2AP Regulates SUMOylation of CIN85 in Podocytes Irini Tossidou,a Rainer Niedenthal,b Malte Klaus,b Beina Teng,a Kirstin Worthmann,a Benjamin L. King,c Kevin J. Peterson,d Hermann Haller,a,c and Mario Schiffera,c Hannover Medical School, Department of Nephrology, Hannover, Germanya; Hannover Medical School, Institute of Physiological Chemistry/Biochemistry, Hannover, Germanyb; Mount Desert Island Biological Laboratory, Salisbury Cove, Maine, USAc; and Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire, USAd Podocytes 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 downregula- tion 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 postrans- lationally 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. he adaptor molecules CD2-associated protein (CD2AP) and Vertebrates contain four ϳ100-amino-acid SUMO proteins, TCbl-interacting protein of 85 kDa (CIN85) belong to a ubiq- SUMO-1, -2, -3, and -4. Of these, SUMO-1 to -3 are ubiquitously uitously expressed protein family of adaptor molecules that are expressed whereas the recently reported SUMO-4 seems to be involved in a variety of cellular processes, like cell signaling (12, 18, expressed mainly in the kidney, lymph node, and spleen. SUMO-2 52), cytoskeletal arrangement (2, 16, 29, 50), and degradative traf- and -3 are nearly identical, whereas SUMO-1 has only 56% iden- ficking and endocytosis of receptors (15, 24, 26, 43, 45, 49, 57). tity with SUMO-2 and -3. SUMOs are similar to ubiquitin in their The two proteins show high sequence and structural similarities, three-dimensional structure, and the steps involved in the SUMO and they both contain three SH3 domains, a proline-rich region, pathway resemble those of the ubiquitin pathway (11, 19). In con- and a coiled-coil domain (7). However, they appear to have com- trast to ubiquitination, SUMOs attach to lysines that are often pletely different functional roles. While CD2AP is solely expressed found within a small consensus motif, ␺KXE (where ␺ is a large in its full-length form, multiple CIN85/Ruk isoforms were iden- hydrophobic amino acid and X can be any amino acid) (41). tified in various tissues and cell lines, due to alternative splicing SUMO modification occurs through an enzymatic pathway and different promoters (3, 31). consisting of an E1 activation enzyme (SAE-2/1), an E2- In podocytes CD2AP is expressed at the slit diaphragm, a spe- conjugating enzyme (Ubc9), and a number of E3 ligases. Ubc9 cialized intercellular junction between neighboring podocytes is capable of directly modifying substrates through interaction covering the outer surface of the glomerular tuft. CD2AP interacts with the SUMO conjugation motif ␺KXE (11, 21). This type of with several proteins at the slit diaphragm. One of the major com- posttranslational modification is an efficient and rapid way of ponents is nephrin, a transmembrane adhesion protein of the Ig controlling the activity of a protein. It is well known that post- superfamily. Humans and mice lacking nephrin are born without translational modifications, such as phosphorylation and ubiq- intact slit diaphragms and develop massive proteinuria in utero uitination, modulate protein interactions (8, 46). There is no (22, 40). Mice deficient in CD2AP are born healthy but develop a simple way to predict what the functional consequence of a rapid-onset nephrotic syndrome at 3 weeks of age and die of renal SUMOylated target will be. One molecular consequence of failure 6 weeks after birth (44). We have previously demonstrated SUMOylation is the inhibition of protein-protein interactions. that deficiency of CD2AP leads to a differentiation-dependent in- An example of this is SUMOylation of C-terminal binding pro- crease of full-length CIN85 expression, which correlates with a tein (CtBP), which loses its interaction with the PDZ domain of loss of expression of the slit diaphragm protein nephrin in podo- nNos (28). SUMOylation can also alter the localization, stabil- cytes. Furthermore, we found that CIN85 is a binding partner of nephrin and that overexpression of CIN85 leads to increased en- docytosis of nephrin after growth factor stimulation (48, 49). Received 10 August 2011 Returned for modification 2 September 2011 Here, we present evidence that CD2AP has a direct influence Accepted 21 December 2011 on posttranslational modification of full-length CIN85. Small Published ahead of print 27 December 2011 ubiquitin-related modifier (SUMO) is a transient and reversible Address correspondence to Irini Tossidou, [email protected], or posttranslational protein modifier that plays an important role in Mario Schiffer, [email protected]. many cellular pathways, including subcellular localization, Copyright © 2012, American Society for Microbiology. All Rights Reserved. protein-protein interaction, transcriptional regulation, activation doi:10.1128/MCB.06106-11 of ion channels, and intracellular localization (11, 35, 38, 56). 1068 mcb.asm.org 0270-7306/12/$12.00 Molecular and Cellular Biology p. 1068–1079 SUMOylation of CIN85 ity, and activity of a protein (11, 35, 38, 56). The ability of The medium consisted of RPMI 1640 (Biochrom, Berlin, Germany) con- CIN85 to bind to other proteins has been attributed to the taining 10% fetal calf serum (FCS) (PAA, Pasching, Austria), 1% phosphorylation status of its binding partners (20, 25, 42). The penicillin-streptomycin (Invitrogen, Carlsbad, CA), and 10 U/ml recom- fact that CIN85 is ubiquitinated (mono-, poly-, and multiubiq- binant mouse gamma interferon (Cell Sciences, Canton, MA). Every ex- perimental setup and result was confirmed in 3 different clones of uitinated) but not degraded by the proteasome has been exten- ϩ/ϩ Ϫ/Ϫ sively studied (14, 51). Ubiquitination is not always associated CD2AP and CD2AP podocytes. Human embryonic kidney (HEK) 293 cells (DSMZ, Braunschweig, Germany) were cultured in medium with the degradation of modified proteins but could also be consisting of Dulbecco’s modified Eagle’s medium (MEM) high glucose involved in regulating the trafficking and enzymatic activities with L-glutamine (PAA, Pasching, Austria) containing 10% fetal bovine of a protein (39). SUMOylation and ubiquitination have also serum (FBS) (PAA, Pasching, Austria) and 1% antibiotic-antimycotic so- been reported to act either sequentially or in concert to regulate lution (PAA, Pasching, Austria). The cells were inoculated every 3 or 4 the function of the substrate protein (4, 17). Until now, it was days into new medium. unknown how the activity and binding ability of CIN85 are Animals. Targeted disruption and generation of CD2AP-deficient ho- regulated. In this study, we demonstrate that CIN85 is post- mozygous mice on a 129/J background is described elsewhere (44). Kid- translationally modified by SUMOylation in the presence of ney tissue and glomeruli were harvested from 4-week-old CD2APϩ/ϩ and Ϫ Ϫ CD2AP and controls its interaction with nephrin. Since we CD2AP / mice. previously demonstrated that CIN85 interaction with nephrin Western blot analysis. To analyze whole-cell protein lysates from cul- induces nephrin ubiquitination and endocytosis
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