The Experts below are selected from a list of 2613 Experts worldwide ranked by ideXlab platform
Guoping Zhou - One of the best experts on this subject based on the ideXlab platform.
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mir 939 5p decreases the enrichment of rna polymerase ii in the promoter region of CD2AP involved in nephrotic syndrome
Journal of Cellular Biochemistry, 2019Co-Authors: Jinya Wang, Daoqi Zhang, Xiaoqin Qiao, Guoping ZhouAbstract:: The expression changes of CD2-associated protein (CD2AP) can lead to kidney diseases with proteinuria, including nephrotic syndrome (NS). A recent study reported that miRNAs may be important transcriptional regulators. In this study, we found increased expression of miR-939-5p and decreased expression of CD2AP in the peripheral blood of patients with NS. However, miR-939-5p did not show a regulatory effect on the 3'-untranslated region of CD2AP. The expression levels of specific protein 1 and adenovirus E2 promoter-binding factor 1, important transcription regulators in the promoter region of CD2AP, were also not affected by microRNA (miR)-939-5p. We confirmed that miR-939-5p is in the nucleus by fluorescent in situ hybridization and cytoplasmic separation polymerase chain reaction. The promoter plasmid and miR-939-5p were cotransfected into HEK-293 cells, and the luciferase reporter gene assay was used to analyze the promoter activity. We found that miR-939-5p binds to a specific sequence in the CD2AP promoter. miR-939-5p was confirmed to reduce the recruitment of RNA polymerase II to the CD2AP promoter region by chromatin immunoprecipitation. These findings improve our understanding of the mechanism of miR-939-5p in NS and provide potential molecular therapeutic targets for NS.
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All- trans retinoic acid up-regulates the human CD2AP gene expression through Sp1/Sp3 binding sites
Immunologic Research, 2015Co-Authors: Huaguo Xu, Guoping ZhouAbstract:All-trans retinoic acid (ATRA), an active metabolite of vitamin A, plays an important role in regulating cell differentiation, proliferation, and apoptosis. It was reported that ATRA could cause an up-regulation of protein expression of CD2AP in nephrotic animals. However, the mechanism of ATRA-mediated up-regulation is not well understood. In the present study, deletion analysis and luciferase assays demonstrated that ATRA caused a marked increase in the activity of the CD2AP promoter, and the region between nt −599 and −328 from the transcription start site, where there are two clusters of Sp1/3 binding sites, was indispensable for ATRA-mediated up-regulation. Chromatin immunoprecipitation assays revealed that ATRA activated the CD2AP transcription through enhancing the DNA-binding activity of Sp1 and Sp3 with the CD2AP promoter. Taken together, this study provided evidence for the first time showing the stimulating effect of ATRA on CD2AP and new therapeutic strategies for the treatment of nephritic syndrome and other associated diseases of CD2AP deficiency.
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all trans retinoic acid up regulates the human CD2AP gene expression through sp1 sp3 binding sites
Immunologic Research, 2015Co-Authors: Huaguo Xu, Guoping ZhouAbstract:All-trans retinoic acid (ATRA), an active metabolite of vitamin A, plays an important role in regulating cell differentiation, proliferation, and apoptosis. It was reported that ATRA could cause an up-regulation of protein expression of CD2AP in nephrotic animals. However, the mechanism of ATRA-mediated up-regulation is not well understood. In the present study, deletion analysis and luciferase assays demonstrated that ATRA caused a marked increase in the activity of the CD2AP promoter, and the region between nt −599 and −328 from the transcription start site, where there are two clusters of Sp1/3 binding sites, was indispensable for ATRA-mediated up-regulation. Chromatin immunoprecipitation assays revealed that ATRA activated the CD2AP transcription through enhancing the DNA-binding activity of Sp1 and Sp3 with the CD2AP promoter. Taken together, this study provided evidence for the first time showing the stimulating effect of ATRA on CD2AP and new therapeutic strategies for the treatment of nephritic syndrome and other associated diseases of CD2AP deficiency.
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transcriptional activation of the human CD2AP promoter by e2f1
PLOS ONE, 2012Co-Authors: Huaguo Xu, Yi Wang, Guoping ZhouAbstract:CD2-associated protein (CD2AP) is an adaptor molecule involved in T cell receptor signaling and podocyte homeostasis. CD2AP-deficient mice develop nephritic syndrome and renal failure caused by glomerulosclerosis. Transcription factor E2F1 is a key regulator of cell proliferation and apoptosis. Here we report that E2F1 up-regulates the human CD2AP promoter and further increases the mRNA and protein levels of the human CD2AP in human embryonic kidney (HEK) 293 cells. By semi-quantitative RT-PCR and Western blot analysis we demonstrate that ectopic expression of E2F1 elevates the mRNA and protein levels of CD2AP. Consistently, transient transfection assays prove that overexpression of E2F1 transactivates the CD2AP promoter while knocking-down of endogenous E2F1 by a shRNA strategy results in reduction of the CD2AP promoter activity. Toward understanding the underlying mechanism of this regulation, we performed chromatin immunoprecipitation and mutations of the putative Sp1 binding sites, demonstrating that E2F1 can bind to Sp1 binding site and overexpression of E2F1 is capable of increasing the binding of E2F1 and decreasing the binding of Sp1 to Sp1 binding sites.
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transcriptional control of human CD2AP expression the role of sp1 and sp3
Molecular Biology Reports, 2012Co-Authors: Huaguo Xu, Yi Wang, Guoping ZhouAbstract:The CD2 associated protein (CD2AP) is characterized as a T-lymphocyte CD2 adapter protein and is found to be related to glomerulosclerosis, and CD2AP knockout mice develop a rapid onset nephrotic syndrome and die of renal failure. Here we report that the transcription factor Sp1 and Sp3 up-regulate the basal transcriptional activity of CD2AP and increase CD2AP expression at mRNA level. We show by Chromatin immunoprecipitation (ChIP) assay that Sp1 and Sp3 interact with the CD2AP promoter region in vivo. By transient transfection analysis we also demonstrate the mutations of Sp1/3 binding sites result in a profound reduction of CD2AP promoter activity. Overexpression of Sp1 and Sp3 transactivates the CD2AP promoter, whereas small interfering RNA-mediated (siRNA) blockage of Sp1 and Sp3 genes expressions inhibits markedly its activity. These results suggest that Sp1 and Sp3 play an important role in regulating CD2AP transcription through binding to the Sp1/3 binding sites.
Andrey S Shaw - One of the best experts on this subject based on the ideXlab platform.
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the adaptor molecule CD2AP in cd4 t cells modulates differentiation of follicular helper t cells during chronic lcmv infection
PLOS Pathogens, 2018Co-Authors: Saravanan Raju, Andrey S Shaw, Kohei Kometani, Tomohiro Kurosaki, Takeshi EgawaAbstract:CD4 T cell-mediated help to CD8 T cells and B cells is a critical arm of the adaptive immune system required for control of pathogen infection. CD4 T cells express cytokines and co-stimulatory molecules that support a sustained CD8 T cell response and also enhance generation of protective antibody by germinal center B cells. However, the molecular components that modulate CD4 T cell functions in response to viral infection or vaccine are incompletely understood. Here we demonstrate that inactivation of the signaling adaptor CD2-associated protein (CD2AP) promotes CD4 T cell differentiation towards the follicular helper lineage, leading to enhanced control of viral infection by augmented germinal center response in chronic lymphocytic choriomeningitis virus (LCMV) infection. The enhanced follicular helper differentiation is associated with extended duration of TCR signaling and enhanced cytokine production of CD2AP-deficient CD4 T cells specifically under TH1 conditions, while neither prolonged TCR signaling nor enhanced follicular helper differentiation was observed under conditions that induce other helper effector subsets. Despite the structural similarity between CD2AP and the closely related adaptor protein CIN85, we observed defective antibody-mediated control of chronic LCMV infection in mice lacking CIN85 in T cells, suggesting non-overlapping and potentially antagonistic roles for CD2AP and CIN85. These results suggest that tuning of TCR signaling by targeting CD2AP improves protective antibody responses in viral infection.
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CD2AP links cortactin and capping protein at the cell periphery to facilitate formation of lamellipodia
Molecular and Cellular Biology, 2013Co-Authors: Jianping Zhao, Serawit Bruck, Saso Cemerski, Lei Zhang, Boyd Butler, Adish Dani, John A Cooper, Andrey S ShawAbstract:Understanding the physiology of complex relationships between components of signaling pathways and the actin cytoskeleton is an important challenge. CD2AP is a membrane scaffold protein implicated in a variety of physiological and disease processes. The physiological function of CD2AP is unclear, but its biochemical interactions suggest that it has a role in dynamic actin assembly. Here, we report that CD2AP functions to facilitate the recruitment of actin capping protein (CP) to the Src kinase substrate, cortactin, at the cell periphery, and that this is necessary for formation of the short branched filaments that characterize lamellipodium formation and are required for cell migration. Superresolution fluorescence microscopy demonstrated that the efficient colocalization of CP and cortactin at the cell periphery required CD2AP. As both cortactin and CP function to enhance branched actin filament formation, CD2AP functions synergistically to enhance the function of both proteins. Our data demonstrate how the interplay between specialized actin regulatory molecules shapes the actin cytoskeleton.
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tβri independently activates smad and CD2AP dependent pathways in podocytes
Journal of The American Society of Nephrology, 2009Co-Authors: Sandhya Xavier, Andrey S Shaw, Mario Schiffer, Thiruvur Niranjan, Stefanie Krick, Taoran Zhang, Wenjun Ju, Erwin P BottingerAbstract:TGF-β regulates differentiation, growth, and apoptosis of podocytes and mediates podocyte depletion in glomerulosclerosis. TGF-β promotes proapoptotic signaling mediated by Smad3 but also activates prosurvival pathways such as phosphoinositide-3 kinase (PI3K)/AKT; the latter requires the CD2-associated adaptor protein (CD2AP) in podocytes. Whether the opposing activities mediated by Smad proteins and CD2AP involve molecular cross-talk is unknown. Here, we report that CD2AP-dependent early activation of the antiapoptotic PI3K/AKT pathway does not require TGF-β receptor–regulated Smad2 and Smad3. We found that the C-terminal region of CD2AP interacts directly with the cytoplasmic tail of the TGF-β receptor type I (TβRI) in a kinase-dependent manner and that the interaction between the TβRI and the p85 subunit of PI3K requires CD2AP. Consistent with the proapoptotic function of Smad signaling, Smad2/3-deficient podocytes were hyperproliferative and resistant to TGF-β–induced growth inhibition and apoptosis. In contrast, CD2AP-deficient cells were hypoproliferative and hypersensitive to TGF-β–induced apoptosis. In vivo , to determine the effects of reduced Smad3 or CD2AP gene dosage on podocyte apoptosis and proteinuria characteristic of TGF-β1 transgenic mice, we generated TGF-β1 transgenic mice deficient for Smad3 or heterozygous for CD2AP. Smad3 deficiency ameliorated podocyte apoptosis, and CD2AP heterozygosity increased both podocyte apoptosis and proteinuria. These data define distinct canonical (Smad) and noncanonical (CD2AP/PI3K/AKT) pathways that arise from direct, independent interactions with the TβRI and that mediate opposing signals for podocyte death or survival.
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CD2AP structure and progression of renal disease
Biophysical Journal, 2009Co-Authors: Brian D Adair, Mehmet M. Altintas, Clemens C. Möller, Jan Flesche, Andrey S Shaw, Christian Faul, Kirk Campbell, Changkyu Gu, Sanja Sever, Peter MundelAbstract:CD2AP is a scaffolding molecule that was originally cloned as an interaction partner of CD2 in T lymphocytes. In the kidney, CD2AP is strongly expressed in podocytes, a cell type that regulates the filtration barrier. The protein directly interacts with filamentous actin and a variety of cell membrane proteins including the kidney filter protein nephrin. In addition to discrete binding sites for actin and nephrin, CD2AP possesses three SH3 domains and a proline-rich region containing, in turn, binding sites for SH3 domains. CD2AP is implicated in dynamic actin remodeling and membrane trafficking that occurs during receptor endocytosis and cytokinesis. We have initiated structural studies of recombinant CD2AP protein using electron microscopy and single particle image analysis. Negative stain electron microscopy of revealed uniform particles with a size and morphology suggesting a tetrameric organization, subsequently verified with chemical crosslinking. Single particle image analysis was used to generate a three-dimensional map of the CD2AP tetramer at 21 A resolution. The electron density map reveals an extended structure allowing the identification of specific subdomains. The tetramer is organized around a central core, including density assigned to the C-terminal coiled-coil domain, surrounded by four loosely attached arms radiating out from the center, which we have assigned to the N-terminal SH3 domains. We have further identified CD2AP as a substrate for cytoplasmic cathepsin L, a protease that is induced in early podocyte damage. Cleavage of CD2AP with cathepsin L results in a C-terminal core domain that is structurally competent but releases the CD2AP binding partner dendrin resulting in translocation of dendrin to the nucleus where it promotes apoptosis. Based on our analysis of the cathepsin L cleavage sites within CD2AP we conclude that cytosolic cathepsin L releases the N-terminal arms producing a structurally competent C-terminal core domain.
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CD2AP cin85 balance determines receptor tyrosine kinase signaling response in podocytes
Journal of Biological Chemistry, 2007Co-Authors: Irini Tossidou, Andrey S Shaw, Christian Kardinal, Imke Peters, Wilhelm Kriz, Ivan Dikic, Sergej Tkachuk, Inna Dumler, Hermann Haller, Mario SchifferAbstract:Abstract Defects in podocyte signaling are the basis of many inherited glomerular diseases leading to glomerulosclerosis. CD2-associated protein (CD2AP) is highly expressed in podocytes and is considered to play an important role in the maintenance of the glomerular slit diaphragm. Mice deficient for CD2AP (CD2AP-/-) appear normal at birth but develop a rapid onset nephrotic syndrome at 3 weeks of age. We demonstrate that impaired intracellular signaling with subsequent podocyte damage is the reason for this delayed podocyte injury in CD2AP-/- mice. We document that CD2AP deficiency in podocytes leads to diminished signal initiation and termination of signaling pathways mediated by receptor tyrosine kinases (RTKs). In addition, we demonstrate that CIN85, a paralog of CD2AP, is involved in termination of RTK signaling in podocytes. CIN85 protein expression is increased in CD2AP-/- podocytes in vitro. Stimulation of CD2AP-/- podocytes with various growth factors, including insulin-like growth factor 1, vascular endothelial growth factor, and fibroblast growth factor, resulted in a significantly decreased phosphatidylinositol 3-kinase/AKT and ERK signaling response. Moreover, increased CIN85 protein is detectable in podocytes in diseased CD2AP-/- mice, leading to decreased base-line activation of ERK and decreased phosphorylation after growth factor stimulation in vivo. Because repression of CIN85 protein leads to a restored RTK signaling response, our results support an important role of CD2AP/CIN85 protein balance in the normal signaling response of podocytes.
Huaguo Xu - One of the best experts on this subject based on the ideXlab platform.
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all trans retinoic acid up regulates the human CD2AP gene expression through sp1 sp3 binding sites
Immunologic Research, 2015Co-Authors: Huaguo Xu, Guoping ZhouAbstract:All-trans retinoic acid (ATRA), an active metabolite of vitamin A, plays an important role in regulating cell differentiation, proliferation, and apoptosis. It was reported that ATRA could cause an up-regulation of protein expression of CD2AP in nephrotic animals. However, the mechanism of ATRA-mediated up-regulation is not well understood. In the present study, deletion analysis and luciferase assays demonstrated that ATRA caused a marked increase in the activity of the CD2AP promoter, and the region between nt −599 and −328 from the transcription start site, where there are two clusters of Sp1/3 binding sites, was indispensable for ATRA-mediated up-regulation. Chromatin immunoprecipitation assays revealed that ATRA activated the CD2AP transcription through enhancing the DNA-binding activity of Sp1 and Sp3 with the CD2AP promoter. Taken together, this study provided evidence for the first time showing the stimulating effect of ATRA on CD2AP and new therapeutic strategies for the treatment of nephritic syndrome and other associated diseases of CD2AP deficiency.
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All- trans retinoic acid up-regulates the human CD2AP gene expression through Sp1/Sp3 binding sites
Immunologic Research, 2015Co-Authors: Huaguo Xu, Guoping ZhouAbstract:All-trans retinoic acid (ATRA), an active metabolite of vitamin A, plays an important role in regulating cell differentiation, proliferation, and apoptosis. It was reported that ATRA could cause an up-regulation of protein expression of CD2AP in nephrotic animals. However, the mechanism of ATRA-mediated up-regulation is not well understood. In the present study, deletion analysis and luciferase assays demonstrated that ATRA caused a marked increase in the activity of the CD2AP promoter, and the region between nt −599 and −328 from the transcription start site, where there are two clusters of Sp1/3 binding sites, was indispensable for ATRA-mediated up-regulation. Chromatin immunoprecipitation assays revealed that ATRA activated the CD2AP transcription through enhancing the DNA-binding activity of Sp1 and Sp3 with the CD2AP promoter. Taken together, this study provided evidence for the first time showing the stimulating effect of ATRA on CD2AP and new therapeutic strategies for the treatment of nephritic syndrome and other associated diseases of CD2AP deficiency.
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transcriptional activation of the human CD2AP promoter by e2f1
PLOS ONE, 2012Co-Authors: Huaguo Xu, Yi Wang, Guoping ZhouAbstract:CD2-associated protein (CD2AP) is an adaptor molecule involved in T cell receptor signaling and podocyte homeostasis. CD2AP-deficient mice develop nephritic syndrome and renal failure caused by glomerulosclerosis. Transcription factor E2F1 is a key regulator of cell proliferation and apoptosis. Here we report that E2F1 up-regulates the human CD2AP promoter and further increases the mRNA and protein levels of the human CD2AP in human embryonic kidney (HEK) 293 cells. By semi-quantitative RT-PCR and Western blot analysis we demonstrate that ectopic expression of E2F1 elevates the mRNA and protein levels of CD2AP. Consistently, transient transfection assays prove that overexpression of E2F1 transactivates the CD2AP promoter while knocking-down of endogenous E2F1 by a shRNA strategy results in reduction of the CD2AP promoter activity. Toward understanding the underlying mechanism of this regulation, we performed chromatin immunoprecipitation and mutations of the putative Sp1 binding sites, demonstrating that E2F1 can bind to Sp1 binding site and overexpression of E2F1 is capable of increasing the binding of E2F1 and decreasing the binding of Sp1 to Sp1 binding sites.
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transcriptional control of human CD2AP expression the role of sp1 and sp3
Molecular Biology Reports, 2012Co-Authors: Huaguo Xu, Yi Wang, Guoping ZhouAbstract:The CD2 associated protein (CD2AP) is characterized as a T-lymphocyte CD2 adapter protein and is found to be related to glomerulosclerosis, and CD2AP knockout mice develop a rapid onset nephrotic syndrome and die of renal failure. Here we report that the transcription factor Sp1 and Sp3 up-regulate the basal transcriptional activity of CD2AP and increase CD2AP expression at mRNA level. We show by Chromatin immunoprecipitation (ChIP) assay that Sp1 and Sp3 interact with the CD2AP promoter region in vivo. By transient transfection analysis we also demonstrate the mutations of Sp1/3 binding sites result in a profound reduction of CD2AP promoter activity. Overexpression of Sp1 and Sp3 transactivates the CD2AP promoter, whereas small interfering RNA-mediated (siRNA) blockage of Sp1 and Sp3 genes expressions inhibits markedly its activity. These results suggest that Sp1 and Sp3 play an important role in regulating CD2AP transcription through binding to the Sp1/3 binding sites.
Peter Mundel - One of the best experts on this subject based on the ideXlab platform.
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CD2AP structure and progression of renal disease
Biophysical Journal, 2009Co-Authors: Brian D Adair, Mehmet M. Altintas, Clemens C. Möller, Jan Flesche, Andrey S Shaw, Christian Faul, Kirk Campbell, Changkyu Gu, Sanja Sever, Peter MundelAbstract:CD2AP is a scaffolding molecule that was originally cloned as an interaction partner of CD2 in T lymphocytes. In the kidney, CD2AP is strongly expressed in podocytes, a cell type that regulates the filtration barrier. The protein directly interacts with filamentous actin and a variety of cell membrane proteins including the kidney filter protein nephrin. In addition to discrete binding sites for actin and nephrin, CD2AP possesses three SH3 domains and a proline-rich region containing, in turn, binding sites for SH3 domains. CD2AP is implicated in dynamic actin remodeling and membrane trafficking that occurs during receptor endocytosis and cytokinesis. We have initiated structural studies of recombinant CD2AP protein using electron microscopy and single particle image analysis. Negative stain electron microscopy of revealed uniform particles with a size and morphology suggesting a tetrameric organization, subsequently verified with chemical crosslinking. Single particle image analysis was used to generate a three-dimensional map of the CD2AP tetramer at 21 A resolution. The electron density map reveals an extended structure allowing the identification of specific subdomains. The tetramer is organized around a central core, including density assigned to the C-terminal coiled-coil domain, surrounded by four loosely attached arms radiating out from the center, which we have assigned to the N-terminal SH3 domains. We have further identified CD2AP as a substrate for cytoplasmic cathepsin L, a protease that is induced in early podocyte damage. Cleavage of CD2AP with cathepsin L results in a C-terminal core domain that is structurally competent but releases the CD2AP binding partner dendrin resulting in translocation of dendrin to the nucleus where it promotes apoptosis. Based on our analysis of the cathepsin L cleavage sites within CD2AP we conclude that cytosolic cathepsin L releases the N-terminal arms producing a structurally competent C-terminal core domain.
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bigenic mouse models of focal segmental glomerulosclerosis involving pairwise interaction of CD2AP fyn and synaptopodin
Journal of Clinical Investigation, 2006Co-Authors: Tobias B Huber, Christopher Kwoh, Peter Mundel, Jeffrey H Miner, Bjorn Hartleben, Hui Wu, Katsuhiko Asanuma, Markus Godel, Ken J Blumer, Andrey S ShawAbstract:: Focal segmental glomerulosclerosis (FSGS) is the most common primary glomerular diagnosis resulting in end-stage renal disease. Defects in several podocyte proteins have been implicated in the etiology of FSGS, including podocin, alpha-actinin-4, CD2-associated protein (CD2AP), and TRPC6. Despite our growing understanding of genes involved in the pathogenesis of focal segmental sclerosis, the vast majority of patients with this disease, even those with a familial linkage, lack a clear genetic diagnosis. Here, we tested whether combinations of genetic heterozygosity (bigenic heterozygosity) that alone do not result in clinical kidney disease could function together to enhance susceptibility to glomerular damage and FSGS. Combinations of CD2AP heterozygosity and heterozygosity of either synaptopodin (Synpo) or Fyn proto-oncogene (Fyn) but not kin of IRRE like 1 (Neph1) resulted in spontaneous proteinuria and in FSGS-like glomerular damage. These genetic interactions were also reflected at a functional level, as we found that CD2AP associates with Fyn and Synpo but not with Neph1. This demonstrates that bigenic heterozygosity can lead to FSGS and suggests that combined mutations in 2 or multiple podocyte genes may be a common etiology for glomerular disease.
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CD2AP localizes to the slit diaphragm and binds to nephrin via a novel c terminal domain
American Journal of Pathology, 2001Co-Authors: Nengyao Shih, Jun Li, Ramzi S Cotran, Peter Mundel, Jeffrey H Miner, Andrey S ShawAbstract:CD2AP, an adapter protein containing multiple SH3 domains, plays a critical role in kidney function. Mice lacking CD2AP die soon after birth because of kidney failure. In the kidney, CD2AP is expressed in glomerular podocytes, which suggests that it may play a role in a specialized adhesion complex known as the slit diaphragm. One of the major components of the slit diaphragm is nephrin, a podocyte-specific protein. Here we demonstrate that CD2AP localizes to the slit diaphragm in podocytes using immunoelectron microscopy and that nephrin and CD2AP co-immunoprecipitate from a podocyte cell line. The specificity of this interaction was verified by mapping studies, which demonstrated that a novel domain at the C terminus of CD2AP interacts with the C-terminal portion of the nephrin cytoplasmic domain. These studies lend further support to the idea that CD2AP plays a role in the structural integrity of the slit diaphragm.
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podocin a raft associated component of the glomerular slit diaphragm interacts with CD2AP and nephrin
Journal of Clinical Investigation, 2001Co-Authors: Karin Schwarz, Andrey S Shaw, Moin A Saleem, Christian Faul, Matias Simons, Jochen Reiser, Wihelm Kriz, Lawrence B Holzman, Peter MundelAbstract:NPHS2 was recently identified as a gene whose mutations cause autosomal recessive steroid-resistant nephrotic syndrome. Its product, podocin, is a new member of the stomatin family, which consists of hairpin-like integral membrane proteins with intracellular NH2- and COOH-termini. Podocin is expressed in glomerular podocytes, but its subcellular distribution and interaction with other proteins are unknown. Here we show, by immunoelectron microscopy, that podocin localizes to the podocyte foot process membrane, at the insertion site of the slit diaphragm. Podocin accumulates in an oligomeric form in lipid rafts of the slit diaphragm. Moreover, GST pull-down experiments reveal that podocin associates via its COOH-terminal domain with CD2AP, a cytoplasmic binding partner of nephrin, and with nephrin itself. That podocin interacts with CD2AP and nephrin in vivo is shown by coimmunoprecipitation of these proteins from glomerular extracts. Furthermore, in vitro studies reveal direct interaction of podocin and CD2AP. Hence, as with the erythrocyte lipid raft protein stomatin, podocin is present in high-order oligomers and may serve a scaffolding function. We postulate that podocin serves in the structural organization of the slit diaphragm and the regulation of its filtration function.
Irini Tossidou - One of the best experts on this subject based on the ideXlab platform.
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tyrosine phosphorylation of CD2AP affects stability of the slit diaphragm complex
Journal of The American Society of Nephrology, 2019Co-Authors: Irini Tossidou, Christian Kardinal, Hermann Haller, Beina Teng, Kirstin Worthmann, Janina Mullerdeile, Tilman Jobstschwan, Patricia Schroder, Patricia Bolanospalmieri, Jonas WillerdingAbstract:BACKGROUND: CD2-associated protein (CD2AP), a slit diaphragm-associated scaffolding protein involved in survival and regulation of the cytoskeleton in podocytes, is considered a "stabilizer" of the slit diaphragm complex that connects the slit diaphragm protein nephrin to the cytoskeleton of the cell. Tyrosine phosphorylation of slit diaphragm molecules can influence their surface expression, but it is unknown whether tyrosine phosphorylation events of CD2AP are also physiologically relevant to slit diaphragm stability. METHODS: We used isoelectric focusing, western blot analysis, and immunofluorescence to investigate phosphorylation of CD2AP, and phospho-CD2AP antibodies and site-directed mutagenesis to define the specific phosphorylated tyrosine residues. We used cross-species rescue experiments in CD2APKD zebrafish and in Drosophila cindrRNAi mutants to define the physiologic relevance of CD2AP phosphorylation of the tyrosine residues. RESULTS: We found that VEGF-A stimulation can induce a tyrosine phosphorylation response in CD2AP in podocytes, and that these phosphorylation events have an important effect on slit diaphragm protein localization and functionality in vivo. We demonstrated that tyrosine in position Y10 of the SH3-1 domain of CD2AP is indispensable for CD2AP function in vivo. We found that the binding affinity of nephrin to CD2AP is significantly enhanced in the absence of Y10; however, unexpectedly, this increased affinity leads not to stabilization but to functional impairment of the glomerular filtration barrier. CONCLUSIONS: Our findings provide insight into CD2AP and its phosphorylation in the context of slit diaphragm functionality, and indicate a fine-tuned affinity balance of CD2AP and nephrin that is influenced by receptor tyrosine kinase stimulation.
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CD2AP regulates sumoylation of cin85 in podocytes
Molecular and Cellular Biology, 2012Co-Authors: Irini Tossidou, Hermann Haller, Beina Teng, Kirstin Worthmann, Rainer Niedenthal, Malte Klaus, Benjamin L King, Kevin J Peterson, Mario SchifferAbstract:: 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 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.
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CD2AP cin85 balance determines receptor tyrosine kinase signaling response in podocytes
Journal of Biological Chemistry, 2007Co-Authors: Irini Tossidou, Andrey S Shaw, Christian Kardinal, Imke Peters, Wilhelm Kriz, Ivan Dikic, Sergej Tkachuk, Inna Dumler, Hermann Haller, Mario SchifferAbstract:Abstract Defects in podocyte signaling are the basis of many inherited glomerular diseases leading to glomerulosclerosis. CD2-associated protein (CD2AP) is highly expressed in podocytes and is considered to play an important role in the maintenance of the glomerular slit diaphragm. Mice deficient for CD2AP (CD2AP-/-) appear normal at birth but develop a rapid onset nephrotic syndrome at 3 weeks of age. We demonstrate that impaired intracellular signaling with subsequent podocyte damage is the reason for this delayed podocyte injury in CD2AP-/- mice. We document that CD2AP deficiency in podocytes leads to diminished signal initiation and termination of signaling pathways mediated by receptor tyrosine kinases (RTKs). In addition, we demonstrate that CIN85, a paralog of CD2AP, is involved in termination of RTK signaling in podocytes. CIN85 protein expression is increased in CD2AP-/- podocytes in vitro. Stimulation of CD2AP-/- podocytes with various growth factors, including insulin-like growth factor 1, vascular endothelial growth factor, and fibroblast growth factor, resulted in a significantly decreased phosphatidylinositol 3-kinase/AKT and ERK signaling response. Moreover, increased CIN85 protein is detectable in podocytes in diseased CD2AP-/- mice, leading to decreased base-line activation of ERK and decreased phosphorylation after growth factor stimulation in vivo. Because repression of CIN85 protein leads to a restored RTK signaling response, our results support an important role of CD2AP/CIN85 protein balance in the normal signaling response of podocytes.
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CD2AP/CIN85 Balance Determines Receptor Tyrosine Kinase Signaling Response in Podocytes
Journal of Biological Chemistry, 2007Co-Authors: Irini Tossidou, Andrey S Shaw, Christian Kardinal, Imke Peters, Wilhelm Kriz, Ivan Dikic, Sergej Tkachuk, Inna Dumler, Hermann Haller, Mario SchifferAbstract:Abstract Defects in podocyte signaling are the basis of many inherited glomerular diseases leading to glomerulosclerosis. CD2-associated protein (CD2AP) is highly expressed in podocytes and is considered to play an important role in the maintenance of the glomerular slit diaphragm. Mice deficient for CD2AP (CD2AP-/-) appear normal at birth but develop a rapid onset nephrotic syndrome at 3 weeks of age. We demonstrate that impaired intracellular signaling with subsequent podocyte damage is the reason for this delayed podocyte injury in CD2AP-/- mice. We document that CD2AP deficiency in podocytes leads to diminished signal initiation and termination of signaling pathways mediated by receptor tyrosine kinases (RTKs). In addition, we demonstrate that CIN85, a paralog of CD2AP, is involved in termination of RTK signaling in podocytes. CIN85 protein expression is increased in CD2AP-/- podocytes in vitro. Stimulation of CD2AP-/- podocytes with various growth factors, including insulin-like growth factor 1, vascular endothelial growth factor, and fibroblast growth factor, resulted in a significantly decreased phosphatidylinositol 3-kinase/AKT and ERK signaling response. Moreover, increased CIN85 protein is detectable in podocytes in diseased CD2AP-/- mice, leading to decreased base-line activation of ERK and decreased phosphorylation after growth factor stimulation in vivo. Because repression of CIN85 protein leads to a restored RTK signaling response, our results support an important role of CD2AP/CIN85 protein balance in the normal signaling response of podocytes.