Kyung Lee

Novel recognition motif on fibroblast growth factor receptor mediates direct association and activation of SNT adapter proteins.

Fibroblast growth factors (FGFs) stimulate tyrosine phosphorylation of a membrane-anchored adapter protein, FRS2/SNT-1, promoting its association with Shp-2 tyrosine phosphatase and upstream activators of Ras. Using the yeast two-hybrid protein-protein interaction assay, we show that FRS2/SNT-1 and a newly isolated SNT-2 protein directly bind to FGF receptor-1 (FGFR-1). A juxtamembrane segment of FGFR-1 and the phosphotyrosine-binding domain of SNTs are both necessary and sufficient for interaction in yeast andin vitro, and FGFR-mediated SNT tyrosine phosphorylationin vivo requires these segments of receptor and SNT. Our findings establish SNTs as direct protein links between FGFR-1 and multiple downstream pathways. The SNT binding motif of FGFR-1 is distinct from previously described phosphotyrosine-binding domain recognition motifs, lacking both tyrosine and asparagine residues.

CONTROL OF CYTOSKELETAL ARCHITECTURE BY THE SRC-SUPPRESSED C KINASE SUBSTRATE, SSECKS

Activation of protein kinase C (PKC) in many cell types results in cytoskeletal reorganization associated with cell proliferation. We previously described a new cell cycle-regulated myristylated PKC substrate, SSeCKS (pronounced essex), that interacts with the actin cytoskeleton [Lin et al., 1995, 1996]. SSeCKS shares significant homology with Gravin, which encodes kinase scaffolding functions for PKC and PKA [Nauert et al., 1997]. This article describes the cellular effects of ectopically expressing SSeCKS in untransformed NIH3T3 fibroblasts. Because the constitutive overexpression of SSeCKS is toxic [Lin et al., 1995], we developed cell lines with tetracycline (tet)-regulated SSeCKS expression. The induction of SSeCKS (removal of tet) caused significant cell flattening and the elaboration of an SSeCKS-associated cortical cytoskeletal matrix resistant to Triton X-100 extraction. Flattened cells were growth-arrested and marked by the formation of cellular projections and the temporary loss of actin stress fibers and vinculin-associated adhesion plaques. SSeCKS overexpression did not affect steady-state levels of actin, vinculin, or focal adhesion kinase (FAK) but did increase integrin-independent FAK tyrosine phosphorylation. Stress fiber loss was coincident with induced SSeCKS expression, strongly suggesting a direct effect. Cytochalasin, and to a lesser extent nocodazole, inhibited SSeCKS-induced cell flattening, however, only cytochalasin affected the shape of pre-flattened cells, suggesting a greater dependence on microfilaments, rather than microtubules. By contrast, only nocodazole caused retraction of the filopodia-like processes. These data indicate a role for SSeCKS in modulating both cytoskeletal and signaling pathways. Thus, we propose to expand SSeCKS scaffolding functions to include the ability to control actin-based cytoskeletal architecture, as well as mitogenic signal pathways.

Structural Basis of SNT PTB Domain Interactions with Distinct Neurotrophic Receptors

SNT adaptor proteins transduce activation of fibroblast growth factor receptors (FGFRs) and neurotrophin receptors (TRKs) to common signaling targets. The SNT-1 phosphotyrosine binding (PTB) domain recognizes activated TRKs at a canonical NPXpY motif and, atypically, binds to nonphosphorylated FGFRs in a region lacking tyrosine or asparagine. Here, using NMR and mutational analyses, we show that the PTB domain utilizes distinct sets of amino acid residues to interact with FGFRs or TRKs in a mutually exclusive manner. The FGFR1 peptide wraps around the beta sandwich structure of the PTB domain, and its binding is possibly regulated by conformational change of a unique C-terminal beta strand in the protein. Our results suggest mechanisms by which SNTs serve as molecular switches to mediate the essential interplay between FGFR and TRK signaling during neuronal differentiation.

Glomerular endothelial cell injury and cross talk in diabetic kidney disease.

Diabetic kidney disease (DKD) remains a leading cause of new-onset end-stage renal disease (ESRD), and yet, at present, the treatment is still very limited. A better understanding of the pathogenesis of DKD is therefore necessary to develop more effective therapies. Increasing evidence suggests that glomerular endothelial cell (GEC) injury plays a major role in the development and progression of DKD. Alteration of the glomerular endothelial cell surface layer, including its major component, glycocalyx, is a leading cause of microalbuminuria observed in early DKD. Many studies suggest a presence of cross talk between glomerular cells, such as between GEC and mesangial cells or GEC and podocytes. PDGFB/PDGFRβ is a major mediator for GEC and mesangial cell cross talk, while vascular endothelial growth factor (VEGF), angiopoietins, and endothelin-1 are the major mediators for GEC and podocyte communication. In DKD, GEC injury may lead to podocyte damage, while podocyte loss further exacerbates GEC injury, forming a vicious cycle. Therefore, GEC injury may predispose to albuminuria in diabetes either directly or indirectly by communication with neighboring podocytes and mesangial cells via secreted mediators. Identification of novel mediators of glomerular cell cross talk, such as microRNAs, will lead to a better understanding of the pathogenesis of DKD. Targeting these mediators may be a novel approach to develop more effective therapy for DKD.

Peroxiredoxin II Restrains DNA Damage-induced Death in Cancer Cells by Positively Regulating JNK-dependent DNA Repair

The 2-Cys peroxiredoxins (Prx) belong to a family of antioxidant enzymes that detoxify reactive oxygen and nitrogen species and are distributed throughout the intracellular and extracellular compartments. However, the presence and role of 2-Cys Prxs in the nucleus have not been studied. This study demonstrates that the PrxII located in the nucleus protects cancer cells from DNA damage-induced cell death. Although the two cytosolic 2-Cys Prxs, PrxI and PrxII, were found in the nucleus, only PrxII knockdown selectively and markedly increased cell death in the cancer cells treated with DNA-damaging agents. The increased death was completely reverted by the nuclearly targeted expression of PrxII in an activity-independent manner. Furthermore, the antioxidant butylated hydroxyanisole did not influence the etoposide-induced cell death. Mechanistically, the knockdown of Prx II expression impaired the DNA repair process by reducing the activation of the JNK/c-Jun pathway. These results suggest that PrxII is likely to be attributed to a tumor survival factor positively regulating JNK-dependent DNA repair with its inhibition possibly sensitizing cancer cells to chemotherapeutic agents.

RTN1 mediates progression of kidney disease by inducing ER stress

Identification of new biomarkers and drug targets for chronic kidney disease (CKD) is required for the development of more effective therapy. Here we report an association between expression of reticulon 1 (RTN1) and severity of CKD. An isoform-specific increase in the expression of RTN1A is detected in the diseased kidneys from mice and humans, and correlates inversely with renal function in patients with diabetic nephropathy. RTN1 overexpression in renal cells induces ER stress and apoptosis, whereas RTN1 knockdown attenuates tunicamycin-induced and hyperglycaemia-induced ER stress and apoptosis. RTN1A interacts with PERK through its N-terminal and C-terminal domains, and mutation of these domains prevents this effect on ER stress. Knockdown of Rtn1a expression in vivo attenuates ER stress and renal fibrosis in mice with unilateral ureteral obstruction, and also attenuates ER stress, proteinuria, glomerular hypertrophy and mesangial expansion in diabetic mice. Together, these data indicate that RTN1A contributes to progression of kidney disease by inducing ER stress.

Peroxiredoxin-2 Represses Melanoma Metastasis by Increasing E-Cadherin/β-Catenin Complexes in Adherens Junctions

In melanoma, transition to the vertical growth phase is the critical step in conversion to a deadly malignant disease. Here, we offer the first evidence that an antioxidant enzyme has a key role in this transition. We found that the antioxidant enzyme peroxiredoxin-2 (Prx2) inversely correlated with the metastatic capacity of human melanoma cells. Silencing Prx2 expression stimulated proliferation and migration, whereas ectopic expression of Prx2 produced the opposite effect. Mechanistic investigations indicated that Prx2 negatively regulated Src/ERK activation status, which in turn fortified adherens junctions function by increasing E-cadherin expression and phospho-Y654-dependent retention of β-catenin in the plasma membrane. In murine melanoma cells, Prx2 silencing enhanced lung metastasis in vivo. Interestingly, the natural compound gliotoxin, which is known to exert a Prx-like activity, inhibited proliferation and migration as well as lung metastasis of Prx2-deficient melanoma cells. Overall, our findings reveal that Prx2 is a key regulator of invasion and metastasis in melanoma, and also suggest a pharmacologic strategy to effectively decrease deadly malignant forms of this disease.

BAMBI elimination enhances alternative TGF-β signaling and glomerular dysfunction in diabetic mice

BMP, activin, membrane-bound inhibitor (BAMBI) acts as a pseudo-receptor for the transforming growth factor (TGF)-β type I receptor family and a negative modulator of TGF-β kinase signaling, and BAMBI−/− mice show mild endothelial dysfunction. Because diabetic glomerular disease is associated with TGF-β overexpression and microvascular alterations, we examined the effect of diabetes on glomerular BAMBI mRNA levels. In isolated glomeruli from biopsies of patients with diabetic nephropathy and in glomeruli from mice with type 2 diabetes, BAMBI was downregulated. We then examined the effects of BAMBI deletion on streptozotocin-induced diabetic glomerulopathy in mice. BAMBI−/− mice developed more albuminuria, with a widening of foot processes, than BAMBI+/+ mice, along with increased activation of alternative TGF-β pathways such as extracellular signal–related kinase (ERK)1/2 and Smad1/5 in glomeruli and cortices of BAMBI−/− mice. Vegfr2 and Angpt1, genes controlling glomerular endothelial stability, were downmodulated in glomeruli from BAMBI−/− mice with diabetes. Incubation of glomeruli from nondiabetic BAMBI+/+ or BAMBI−/− mice with TGF-β resulted in the downregulation of Vegfr2 and Angpt1, effects that were more pronounced in BAMBI−/− mice and were prevented by a MEK inhibitor. The downregulation of Vegfr2 in diabetes was localized to glomerular endothelial cells using a histone yellow reporter under the Vegfr2 promoter. Thus, BAMBI modulates the effects of diabetes on glomerular permselectivity in association with altered ERK1/2 and Smad1/5 signaling. Future therapeutic interventions with inhibitors of alternative TGF-β signaling may therefore be of interest in diabetic nephropathy.

Inactivation of Integrin-β1 Prevents the Development of Polycystic Kidney Disease after the Loss of Polycystin-1

Dysregulation of polycystin-1 (PC1) leads to autosomal dominant polycystic kidney disease (ADPKD), a disorder characterized by the formation of multiple bilateral renal cysts, the progressive accumulation of extracellular matrix (ECM), and the development of tubulointerstitial fibrosis. Correspondingly, cystic epithelia express higher levels of integrins (ECM receptors that control various cellular responses, such as cell proliferation, migration, and survival) that are characteristically altered in cystic cells. To determine whether the altered expression of ECM and integrins could establish a pathologic autostimulatory loop, we tested the role of integrin-β1 in vitro and on the cystic development of ADPKD in vivo. Compared with wild-type cells, PC1-depleted immortalized renal collecting duct cells had higher levels of integrin-β1 and fibronectin and displayed increased integrin-mediated signaling in the presence of Mn(2+). In mice, conditional inactivation of integrin-β1 in collecting ducts resulted in a dramatic inhibition of Pkd1-dependent cystogenesis with a concomitant suppression of fibrosis and preservation of normal renal function. Our data provide genetic evidence that a functional integrin-β1 is required for the early events leading to renal cystogenesis in ADPKD and suggest that the integrin signaling pathway may be an effective therapeutic target for slowing disease progression.

Cilium, centrosome and cell cycle regulation in polycystic kidney disease

Polycystic kidney disease is the defining condition of a group of common life-threatening genetic disorders characterized by the bilateral formation and progressive expansion of renal cysts that lead to end stage kidney disease. Although a large body of information has been acquired in the past years about the cellular functions that characterize the cystic cells, the mechanisms triggering the cystogenic conversion are just starting to emerge. Recent findings link defects in ciliary functions, planar cell polarity pathway, and centrosome integrity in early cystic development. Many of the signals dysregulated during cystogenesis may converge on the centrosome for its central function as a structural support for cilia formation and a coordinator of protein trafficking, polarity, and cell division. Here, we will discuss the contribution of proliferation, cilium and planar cell polarity to the cystic signal and will analyze in particular the possible role that the basal bodies/centrosome may play in the cystogenetic mechanisms. This article is part of a Special Issue entitled: Polycystic Kidney Disease.