CD2相关蛋白在足细胞分化中的作用

本文旨在研究肾脏足细胞的分化特点及CD2相关蛋白(CD2-associated protein,CD2AP)在足细胞分化过程中的作用.用RPMI 1640培养基在33.C许可条件下培养永生化小鼠足细胞系(未分化组),转染针对CD2AP的小分子干扰RNA(smallinterfering RNA,siRNA)后置于37.C非许可条件下培养(转染组),并将非许可条件下未转染组作为对照组.用MTT法检测足细胞的生长速度;用RT-PCR方法检测CD2AP、WTI、synaptopodin和nephrin mRNA表达;用Western blot检测CD2AP、wTl和nephrin蛋白表达;用免疫荧光结合激光共聚焦方法检测CD2AP、nephrin、F-actin和tubulin在分化及未分化足细胞中的分布及其共定位情况.结果显示,CD2AP、WTl和nephrin在分化及未分化足细胞中均可稳定表达,而synaptopodin仅表达于已分化足细胞,在未分化足细胞无表达.在足细胞分化过程中,CD2AP和nephrin的表达上调(P<0.05);CD2AP、tubulin和F-actin在细胞内的分布发生改变,CD2AP与nephrin及F-actin在未分化足细胞中存在共定位关系.转染特异性siRNA下调CD2AP表达,细胞生长速度明显减慢,synaptopodin mRNA表达下调(P<0.05),细胞分化迟滞.结果表明,足细胞分化过程中伴随细胞骨架的重新分布和细胞形态的改变;CD2AP可能作为足细胞裂孔隔膜分子与细胞骨架的连接蛋白,在足细胞分化过程中发挥重要作用.     

CD2-associated protein (CD2AP) expression in podocytes rescues lethality of CD2AP deficiency

Mice born without CD2-associated protein (CD2AP) develop renal failure and nephrotic syndrome about 4 weeks after birth and die around 6 weeks of age. Although CD2AP is widely expressed, the severity of the renal failure precludes a clear determination of the role of CD2AP in other tissues. Here we generated transgenic mice expressing CD2AP using a podocyte-specific promoter. Podocyte-specific expression of CD2AP prevented the development of proteinuria, demonstrating that the renal failure is solely due to loss of CD2AP in podocytes and not in other renal or in immune cells. CD2AP-deficient mice are long-lived and appear phenotypically normal. Histological analysis demonstrated testicular abnormalities that were age-related. CIN85, a paralog of CD2AP, is poorly expressed in both the podocyte and the basal seminiferous tubule, suggesting that the loss of CD2AP in specific tissues may be compensated for by CIN85.     

Role of CD2-associated protein in albumin overload-induced apoptosis in podocytes

Proteinuria is a well-established exacerbating factor of chronic kidney diseases. However, the harmful effects of protein overload on podocytes and the underlying mechanisms are still poorly understood. In the present study, we examined the effects of high concentrations of albumin on podocytes and investigated the role of CD2AP (CD2-associated protein) in albumin overload-induced podocyte apoptosis. Conditionally immortalized mouse podocytes were cultured in vitro and treated with different concentrations of BSA. In addition, CD2AP eukaryotic expression vector or siRNA (small interfering RNA) was transfected into podocytes before they were exposed to BSA. Podocyte apoptosis, expressions of active caspase-3 (p17) and CD2AP, and the distribution of F-actin cytoskeleton were detected by flow cytometry, Western-blot analysis and fluorescent staining respectively. It was found that exposure of podocytes to BSA induced podocyte apoptosis in a concentration-dependent manner that was accompanied by up-regulation of active caspase-3, the disruption of F-actin cytoskeleton, and decreased expression of CD2AP. Transfection of CD2AP eukaryotic expression vector into podocytes increased CD2AP expression, partially restored F-actin distribution, blocked active caspase-3 expression and inhibited podocyte apoptosis. In contrast, transfection of CD2AP siRNA deteriorated the above changes induced by BSA. It is concluded that protein overload induces podocyte apoptosis via the down-regulation of CD2AP and subsequent disruption of cytoskeleton of podocytes, and CD2AP may play an important role in protein overload-induced podocyte injury.     

Identification of a novel inhibitory actin-capping protein binding motif in CD2-associated protein

CD2-associated protein (CD2AP) is a scaffold molecule that plays a critical role in the maintenance of the kidney filtration barrier. Little, however, is understood about its mechanism of function. We used mass spectrometry to identify CD2AP-interacting proteins. Many of the proteins that we identified suggest a role for CD2AP in endocytosis and actin regulation. To address the role of CD2AP in regulation of the actin cytoskeleton, we focused on characterizing the interaction of CD2AP with actin-capping protein CP. We identified a novel binding motif LXHXTXXRPK(X)6P present in CD2AP that is also found in its homolog Cin85 and other capping protein-associated proteins such as CARMIL and CKIP-1. CD2AP inhibits the function of capping protein in vitro. Therefore, our results support a role of CD2AP in the regulation of the actin cytoskeleton.     

Downregulation of CD2-associated protein impaired the physiological functions of podocytes

Emerging evidences show that CD2-associated protein (CD2AP) is involved in podocyte injury and the pathogenesis of proteinuria. However, the exact molecular mechanism by which CD2AP exerts its biological function is elusive. We knocked down CD2AP gene by target siRNA in conditionally immortalized mouse podocytes, which showed lowered cell adhesion and spreading ability (P < 0.05). At the same time, cell cycle was arrested in G2/M phase (P < 0.05), and pathologic nuclear division could easily be seen in CD2AP siRNA-transfected podocytes. The proliferation of podocytes were also inhibited significantly by CD2AP siRNA transfection (P < 0.05). Further study revealed disordered distributions of F-actin, as well as lowered nephrin expression and phosphorylation in podocytes. These data suggest that CD2AP may play a crucial role in maintaining the normal function of podocytes and lowered CD2AP causes podocyte injury by disrupting the cytoskeleton and disturbing the nephrin-CD2AP signaling pathway.     

CD2-associated protein is widely expressed and differentially regulated during embryonic development

CD2-associated protein (CD2AP) is an adapter protein that is involved in various signaling and vesicular trafficking processes and also functions as a linker between plasma membrane proteins and the actin cytoskeleton. The protein is known to have important functions in T cells and glomerular podocytes, but it is also expressed by many other adult-type tissues and cells. Here we analyzed the expression of the protein during early embryonic development and organogenesis of the mouse. The results showed differential tissue-specific regulation of CD2AP in developing and maturing organs. In oocytes and pre-implantation embryos, CD2AP was located diffusely in the cytoplasm, whereas in late blastocysts it was concentrated to the intercellular contacts. During organogenesis, CD2AP was distinctly upregulated upon, e.g., the pretubular aggregation of metanephric mesenchyme cells and the appearance of the osteoblastic rim around cartilages during endochondral ossification. High CD2AP expression was also observed during epithelial-like conversion of some highly specialized secretory cell types such as the odontoblasts, the cells of the choroid plexus and the decidualized cells of the endometrial stroma. In other instances, such as the development of the proximal tubuli of the kidney and the flat alveolar epithelium of the lung, the protein was downregulated upon differentiation and maturation of the cells. Finally, certain cells, e.g., glomerular podocytes, those forming the collecting ducts of the kidney, and the urothelium of the kidney pelvis, expressed CD2AP throughout their differentiation and maturation. Multiple molecules and complex pathways regulate embryogenesis, and scaffolding proteins apparently have pivotal roles in targeting and finetuning, e.g., growth factor- or hormone-induced processes. The cell-type specific spatio-temporal regulation of CD2AP during development suggests that this adapter protein is a key regulatory partner in many signaling pathways and cellular processes governing differentiation and morphogenesis.     

In vivo interaction of the adapter protein CD2-associated protein with the type 2 polycystic kidney disease protein, polycystin-2

We identified a developmentally regulated gene from mouse kidney whose expression is up-regulated in metanephrogenic mesenchyme cells when they are induced to differentiate to epithelial cells during kidney organogenesis. The deduced 70.5-kDa protein, originally named METS-1 (mesenchyme-to-epithelium transition protein with SH3 domains), has since been cloned as a CD2-associated protein (CD2AP). CD2AP is strongly expressed in glomerular podocytes, and the absence of CD2AP in mice results in congenital nephrotic syndrome. We have found that METS-1/CD2AP (hereafter referred to as CD2AP) is expressed at lower levels in renal tubular epithelial cells in the adult kidney, particularly in distal nephron segments. Independent yeast two-hybrid screens using the COOH-terminal region of either CD2AP or polycystin-2 as bait identified the COOH termini of polycystin-2 and CD2AP, respectively, as strong interacting partners. This interaction was confirmed in cultured cells by co-immunoprecipitation of endogenous polycystin-2 with endogenous CD2AP and vice versa. CD2AP shows a diffuse reticular cytoplasmic and perinuclear pattern of distribution, similar to polycystin-2, in cultured cells, and the two proteins co-localize by indirect double immunofluorescence microscopy. CD2AP is an adapter molecule that associates with a variety of membrane proteins to organize the cytoskeleton around a polarized site. Such a function fits well with that hypothesized for the polycystin proteins in renal tubular epithelial cells, and the present findings suggest that CD2AP has a role in polycystin-2 function.     

A novel role for the adaptor molecule CD2-associated protein in transforming growth factor-beta-induced apoptosis

CD2-associated protein (CD2AP) is an adaptor molecule involved in T cell receptor signaling and podocyte homeostasis. CD2AP-deficient mice develop nephrotic syndrome and renal failure caused by glomerulosclerosis. Here we report that increased transforming growth factor-beta1 (TGF-beta1) expression and apoptosis were present in podocytes at the onset of albuminuria and were followed by depletion of podocytes associated with progressive focal-segmental glomerulosclerosis in CD2AP-/- mice. Conditionally immortalized podocytes derived from CD2AP-/- mice were more susceptible to TGF-beta-induced apoptosis compared with CD2AP+/+ podocytes. Reconstitution of CD2AP rescued CD2AP-/- podocytes from TGF-beta-induced apoptosis. CD2AP was required for early activation of anti-apoptotic phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase 1/2 by TGF-beta. In contrast, activation of pro-apoptotic p38 MAPK by TGF-beta was accelerated and enhanced in the absence of CD2AP. CD2AP was not required for PI3K/AKT activation by insulin and epidermal growth factor, indicating that CD2AP is a selective mediator of anti-apoptotic TGF-beta signaling. In summary, we identified CD2AP as a novel mediator for selective activation of survival pathways and repression of apoptosis signaling by TGF-beta in podocytes. Together, our in vitro and in vivo findings suggest that TGF-beta-induced podocyte apoptosis is an early pathomechanism in mice developing focal-segmental glomerulosclerosis associated with functional impairment of CD2AP.     

Regulation of CD2-associated protein influences podocyte endoplasmic reticulum stress-mediated apoptosis induced by albumin overload

The adaptation of microorganisms to pesticide biodegradation relies on the recruitment of catabolic genes by horizontal gene transfer and homologous recombination mediated by insertion sequences (IS). This environment-friendly function is maintained in the degrading population but it has a cost which could diminish its fitness. The loss of genes in the course of evolution being a major mechanism of ecological specialization, we mimicked evolution in vitro by sub-culturing the atrazine-degrading Pseudomonas sp. ADP in a liquid medium containing cyanuric acid as the sole source of nitrogen. After 120 generations, a new population evolved, which replaced the original one. This new population grew faster on cyanuric acid but showed a similar cyanuric acid degrading ability. Plasmid profiles and Southern blot analyses revealed the deletion of a 47 kb region from pADP1 containing the atzABC genes coding for the enzymes that turn atrazine into cyanuric acid. Long PCR and sequencing analyses revealed that this deletion resulted from a homologous recombination between two direct repeats of a 110-bp, identical to ISPps1 of Pseudomonas huttiensis, flanking the deleted 47 kb region. The loss of a region containing three functional genes constitutively expressed thereby constituting a genetic burden under cyanuric acid selection pressure was responsible for the gain in fitness of the new population. It highlights the IS-mediated plasticity of the pesticide-degrading potential and shows that IS not only favours the expansion of the degrading genetic potential thanks to dispersion and duplication events but also contribute to its reduction thanks to deletion events.     

CD2-associated protein/phosphoinositide 3-kinase signaling has a preventive role in angiotensin II-induced podocyte apoptosis

Angiotensin II (Ang II) works as a paracrine or autocrine cytokine agent to regulate renal functions and promotes podocytes dysfunction directly or indirectly, causing proteinuria. The glomerular slit diaphragm (SD) serves as a size-selective barrier and is linked to the actin-based cytoskeleton by adaptor proteins, including CD2-associated protein (CD2AP). Therefore, damages to CD2AP affect not only the function of the SD, but also directly disrupt the podocyte cytoskeleton, leading to proteinuria. In addition, CD2AP can facilitate the nephrin-induced phosphoinositide 3-kinase (PI3-K)/Akt signaling, which protects podocytes from apoptosis. Here we found that CD2AP staining was located diffusely but predominantly in the peripheral cytoplasm and CD2AP co-localized with nephrin in mouse podocytes; however, Ang II decreased CD2AP staining diffusely and induced a separation from concentrated nephrin. Ang II notably reduced CD2AP expression in time- and concentration-dependent manners, and this was significantly recovered by losartan. Ang II induced podocyte apoptosis in time- and concentration-dependent manners in TUNEL and FACS assays. LY294002, a PI3-K inhibitor, further reduced CD2AP expression and increased podocyte apoptosis, which was augmented by siRNA for CD2AP. Thus, Ang II induces the relocalization and reduction of CD2AP via AT1R, which would cause podocyte apoptosis by the suppression of CD2AP/PI3-K signaling.     

Functional involvement of human discs large tumor suppressor in cytokinesis

Cytokinesis is the final step of cell division that completes the separation of two daughter cells. We found that the human discs large (hDlg) tumor suppressor homologue is functionally involved in cytokinesis. The guanylate kinase (GUK) domain of hDlg mediates the localization of hDlg to the midbody during cytokinesis, and over-expression of the GUK domain in U2OS and HeLa cells impaired cytokinesis. Mouse embryonic fibroblasts (MEFs) derived from dlg mutant mice contained an increased number of multinucleated cells and showed reduced proliferation in culture. A kinesin-like motor protein, GAKIN, which binds directly to the GUK domain of hDlg, exhibited a similar intracellular distribution pattern with hDlg throughout mitosis and localized to the midbody during cytokinesis. However, the targeting of hDlg and GAKIN to the midbody appeared to be independent of each other. The midbody localization of GAKIN required its functional kinesin-motor domain. Treatment of cells with the siRNA specific for hDlg and GAKIN caused formation of multinucleated cells and delayed cytokinesis. Together, these results suggest that hDlg and GAKIN play functional roles in the maintenance of midbody architecture during cytokinesis.     

Interactions of CD45-associated protein with the antigen receptor signaling machinery in T-lymphocytes.

CD45 is a transmembrane protein tyrosine phosphatase playing an essential role during T-cell activation. This function relates to the ability of CD45 to regulate p56(lck), a cytoplasmic protein tyrosine kinase necessary for T-cell antigen receptor (TCR) signaling. Previous studies have demonstrated that CD45 is constitutively associated in T-lymphocytes with a transmembrane molecule termed CD45-AP (or lymphocyte phosphatase-associated phosphoprotein). Even though the exact role of this polypeptide is unclear, recent analyses of mice lacking CD45-AP have indicated that its expression is also required for optimal T-cell activation. Herein, we wished to understand better the function of CD45-AP. The results of our studies showed that in T-cells, CD45-AP is part of a multimolecular complex that includes not only CD45, but also TCR, the CD4 and CD8 coreceptors, and p56(lck). The association of CD45-AP with TCR, CD4, and CD8 seemed to occur via the shared ability of these molecules to bind CD45. However, binding of CD45-AP to p56(lck) could take place in the absence of other lymphoid-specific components, suggesting that it can be direct. Structure-function analyses demonstrated that such an interaction was mediated by an acidic segment in the cytoplasmic region of CD45-AP and by the kinase domain of p56(lck). Interestingly, the ability of CD45-AP to interact with Lck in the absence of other lymphoid-specific molecules was proportional to the degree of catalytic activation of p56(lck). Together, these findings suggest that CD45-AP is an adaptor molecule involved in orchestrating interactions among components of the antigen receptor signaling machinery. Moreover, they raise the possibility that one of the functions of CD45-AP is to recognize activated Lck molecules and bring them into the vicinity of CD45.     

FPRP, a major, highly stoichiometric, highly specific CD81- and CD9-associated protein

CD81 and CD9, members of the transmembrane-4 superfamily (TM4SF; tetraspanins), form extensive complexes with other TM4SF proteins, integrins, and other proteins, especially in mild detergents. In moderately stringent Brij 96 lysis conditions, CD81 and CD9 complexes are virtually identical to each other, but clearly distinct from other TM4SF complexes. One of the most prominent proteins within CD81 and CD9 complexes is identified here as FPRP, the 133-kDa prostaglandin F(2alpha) receptor regulatory protein. FPRP, a cell-surface Ig superfamily protein, associates specifically with CD81 or with CD81 and CD9, but not with integrins or other TM4SF proteins. In contrast to other CD81- and CD9-associating proteins, FPRP associates at very high stoichiometry, with essentially 100% of cell-surface FPRP on 293 cells being CD81- and CD9-associated. Also, CD81.CD9.FPRP complexes have a discrete size (<4 x 10(6) Da) as measured by gel permeation chromatography and remain intact after disruption of cholesterol-rich membrane microdomains by methyl-beta-cyclodextrin. Although CD81 associated with both alpha(3) integrin and FPRP in 293 cells, the alpha(3)beta(1).CD81 and CD81.CD9.FPRP complexes were distinct, as determined by immunoprecipitation and immunodepletion experiments. In conclusion, our data affirm the existence of distinct TM4SF complexes with unique compositions and specifically characterize FPRP as the most robust, highly stoichiometric CD81- and/or CD9-associated protein yet described.     

Conditional mutations in gamma-tubulin reveal its involvement in chromosome segregation and cytokinesis

gamma-Tubulin is a conserved essential protein required for assembly and function of the mitotic spindle in humans and yeast. For example, human gamma-tubulin can replace the gamma-tubulin gene in Schizosaccharomyces pombe. To understand the structural/functional domains of gamma-tubulin, we performed a systematic alanine-scanning mutagenesis of human gamma-tubulin (TUBG1) and studied phenotypes of each mutant allele in S. pombe. Our screen, both in the presence and absence of the endogenous S. pombe gamma-tubulin, resulted in 11 lethal mutations and 12 cold-sensitive mutations. Based on structural mapping onto a homology model of human gamma-tubulin generated by free energy minimization, all deleterious mutations are found in residues predicted to be located on the surface, some in positions to interact with alpha- and/or beta-tubulins in the microtubule lattice. As expected, one class of tubg1 mutations has either an abnormal assembly or loss of the mitotic spindle. Surprisingly, a subset of mutants with abnormal spindles does not arrest in M phase but proceeds through anaphase followed by abnormal cytokinesis. These studies reveal that in addition to its previously appreciated role in spindle microtubule nucleation, gamma-tubulin is involved in the coordination of postmetaphase events, anaphase, and cytokinesis.     

A Cortactin-CD2-associated protein (CD2AP) complex provides a novel link between epidermal growth factor receptor endocytosis and the actin cytoskeleton

Growth factor regulation of the cortical actin cytoskeleton is fundamental to a wide variety of cellular processes. The cortical actin-associated protein, cortactin, regulates the formation of dynamic actin networks via the actin-related protein (Arp)2/3 complex and hence is a key mediator of such responses. In order to reveal novel roles for this versatile protein, we used a proteomics-based approach to isolate cortactin-interacting proteins. This identified several proteins, including CD2-associated protein (CD2AP), as targets for the cortactin Src homology 3 domain. Co-immunoprecipitation of CD2AP with cortactin occurred at endogenous expression levels, was transiently induced by epidermal growth factor (EGF) treatment, and required the cortactin Src homology 3 domain. The CD2AP-binding site for cortactin mapped to the second of three proline-rich regions. Because CD2AP is closely related to Cbl-interacting protein of 85 kDa (CIN85), which regulates growth factor receptor down-regulation via complex formation with Cbl and endophilin, we investigated whether the CD2AP-cortactin complex performs a similar function. EGF treatment of cells led to transient association of Cbl and the epidermal growth factor receptor (EGFR) with a constitutive CD2AP-endophilin complex. Cortactin was recruited into this complex with slightly delayed kinetics compared with Cbl and the EGFR. Immunofluorescence analysis revealed that the EGFR, CD2AP, and cortactin co-localized in regions of EGF-induced membrane ruffles. Therefore, by binding both CD2AP and the Arp2/3 complex, cortactin links receptor endocytosis to actin polymerization, which may facilitate the trafficking of internalized growth factor receptors.