Distinct Requirements for Adaptor Proteins NCK1 and NCK2 in Mammary Gland Development.

The adaptor proteins NCK1 and NCK2 are well-established signalling nodes that regulate diverse biological processes including cell proliferation and actin dynamics in many tissue types. Here we have investigated the distribution and function of Nck1 and Nck2 in the developing mouse mammary gland. Using publicly available single-cell RNA sequencing data, we uncovered distinct expression profiles between the two paralogs. Nck1 showed widespread expression in luminal, basal, stromal and endothelial cells, while Nck2 was restricted to luminal and basal cells, with prominent enrichment in hormone-sensing luminal subtypes. Next, using mice with global knockout of Nck1 or Nck2, we assessed mammary gland development during and after puberty (5, 8 and 12 weeks of age). Mice lacking Nck1 or Nck2 displayed significant defects in ductal outgrowth and branching at 5 weeks compared to controls, and the defects persisted in Nck2 knockout mice at 8 weeks before normalizing at 12 weeks. These defects were accompanied by an increase in epithelial cell proliferation at 5 weeks and a decrease at 8 weeks in both Nck1 and Nck2 knockout mice. We also profiled expression of several key genes associated with mammary gland development at these timepoints and detected temporal changes in transcript levels of hormone receptors as well as effectors of cell proliferation and migration in Nck1 and Nck2 knockout mice, in line with the distinct phenotypes observed at 5 and 8 weeks. Together these studies reveal a requirement for NCK proteins in mammary gland morphogenesis, and suggest that deregulation of Nck expression could drive breast cancer progression and metastasis.

Sex Differences in Glomerular Protein Expression and Effects of Soy-Based Diet on Podocyte Signaling.

Background: Kidney disease is a major public health issue arising from loss of glomerular podocyte function, and there are considerable sex differences in its prognosis. Evidence suggests a renoprotective effect of estrogen and soy diet-derived phytoestrogens, although the molecular basis for this is poorly understood. Objective: Here, we aim to assess sex differences in expression of key proteins associated with podocyte survival and determine the effects of dietary soy on glomerular and podocyte signaling. Methods: Male and female FVB mice were fed control, low (1%), and high (20%) doses of isolated soy protein (ISP) in utero and until 100 days of age. Spot urine was collected to measure proteinuria and isolated glomeruli were used to quantify activated and total levels of nephrin, Akt, and ERK1/2. To investigate protective effects of specific soy phytoestrogens, cultured podocytes were treated with or without daidzein and subject to control or high glucose as a model of podocyte injury. Results: Nephrin and Akt were elevated at baseline in glomeruli from females compared to males. Both sexes that were fed 1% and 20% ISP displayed robust increases in total glomerular Akt compared to controls, and these effects were more prominent in females. A similar trend at both doses in both sexes was observed with activated Akt and total nephrin. Notably, males exclusively showed increased phosphorylation of nephrin and extracellular signal-regulated kinase (ERK) at the 1% ISP dose; however, no overt changes in urinary albumin excretion or podocin levels were observed, suggesting that the soy diets did not impair podocyte function. Finally, in cultured male and female podocytes, daidzein treatment suppressed high glucose-induced ERK activation. Conclusions: Together, our findings reveal a putative mechanism to explain the protective influence of sex on kidney disease progression, and they provide further evidence to support a beneficial role for dietary soy in preserving glomerular function.

Contexte: L'insuffisance rénale est un problème majeur de santé publique résultant d'une perte de fonction des podocytes glomérulaires, et son pronostic diffère selon le sexe. Bien que le fondement moléculaire en soit mal compris, des données suggèrent que les œstrogènes et des phytoestrogènes dérivés du soja alimentaire auraient un effet néphroprotecteur. Objectifs: Évaluer les différences selon le sexe dans l'expression des protéines clés associées à la survie des podocytes, et déterminer les effets du soja alimentaire sur la signalisation glomérulaire et les podocytaire. Méthodologie: Des souris FVB mâles et femelles ont reçu un régime alimentaire témoin ou un regime à faible dose (1 %) ou à dose élevée (20 %) de protéines de soja isolées (PSI) in utero et jusqu'à l'âge de 100 jours. Des échantillons aléatoires d'urine ont été recueillis pour mesurer la protéinurie et des glomérules isolés ont été utilisés pour quantifier les niveaux activés et totaux de néphrine, d'Akt et d'ERK1/2. Pour évaluer l'effet protecteur de certains phytoestrogènes du soja, des podocytes cultivés ont été traités avec ou sans daidzéine et soumis à une dose témoin ou à une dose élevée de glucose comme modèle de lésion podocytaire. Résultats: Les taux initiaux de néphrine et d'Akt étaient plus élevés dans les glomérules des souris femelles. Les souris mâles et femelles nourries avec des doses de 1 % et de 20 % de PSI ont montré des augmentations significatives de l'Akt glomérulaire totale par rapport aux témoins, et ces effets étaient plus importants chez les femelles. Une tendance semblable a été observée chez les deux sexes et pour les deux doses en ce qui concerne l'Akt activée et la néphrine totale. Seuls les mâles ont montré une augmentation de la phosphorylation de la néphrine et de l'ERK à 1 % de PSI; aucun changement manifeste n'a cependant été observé dans l'excrétion urinaire d'albumine ou dans le taux de podocine, ce qui suggère que le soja alimentaire n'a pas altéré la fonction des podocytes. Dans les podocytes cultivés, tant mâles que femelles, le traitement à la daidzéine a inhibé l'activation de l'ERK induite par une forte dose de glucose. Conclusion: Ensemble, nos résultats révèlent un mécanisme putatif pouvant expliquer l'effet protecteur du sexe du patient sur la progression de l'insuffisance rénale. Ces résultats fournissent des preuves supplémentaires soutenant l'hypothèse d'un rôle bénéfique du soja alimentaire dans la préservation de la fonction glomérulaire.

Complementary Nck1/2 Signaling in Podocytes Controls α Actinin-4-Mediated Actin Organization, Adhesion, and Basement Membrane Composition.

BACKGROUND: Maintenance of the kidney filtration barrier requires coordinated interactions between podocytes and the underlying glomerular basement membrane (GBM). GBM ligands bind podocyte integrins, which triggers actin-based signaling events critical for adhesion. Nck1/2 adaptors have emerged as essential regulators of podocyte cytoskeletal dynamics. However, the precise signaling mechanisms mediated by Nck1/2 adaptors in podocytes remain to be fully elucidated. METHODS: We generated podocytes deficient in Nck1 and Nck2 and used transcriptomic approaches to profile expression differences. Proteomic techniques identified specific binding partners for Nck1 and Nck2 in podocytes. We used cultured podocytes and mice deficient in Nck1 and/or Nck2, along with podocyte injury models, to comprehensively verify our findings. RESULTS: Compound loss of Nck1/2 altered expression of genes involved in actin binding, cell adhesion, and extracellular matrix composition. Accordingly, Nck1/2-deficient podocytes showed defects in actin organization and cell adhesion in vitro, with podocyte detachment and altered GBM morphology present in vivo. We identified distinct interactomes for Nck1 and Nck2 and uncovered a mechanism by which Nck1 and Nck2 cooperate to regulate actin bundling at focal adhesions via α actinin-4. Furthermore, loss of Nck1 or Nck2 resulted in increased matrix deposition in vivo, with more prominent defects in Nck2-deficient mice, consistent with enhanced susceptibility to podocyte injury. CONCLUSION: These findings reveal distinct, yet complementary, roles for Nck proteins in regulating podocyte adhesion, controlling GBM composition, and sustaining filtration barrier integrity.

Receptor Tyrosine Kinase Signaling and Targeting in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is amongst the deadliest of human cancers, with a median survival rate of just over one year following diagnosis. Characterized by rapid proliferation and diffuse infiltration into the brain, GBM is notoriously difficult to treat, with tumor cells showing limited response to existing therapies and eventually developing resistance to these interventions. As such, there is intense interest in better understanding the molecular alterations in GBM to guide the development of more efficient targeted therapies. GBM tumors can be classified into several molecular subtypes which have distinct genetic signatures, and they show aberrant activation of numerous signal transduction pathways, particularly those connected to receptor tyrosine kinases (RTKs) which control glioma cell growth, survival, migration, invasion, and angiogenesis. There are also non-canonical modes of RTK signaling found in GBM, which involve G-protein-coupled receptors and calcium channels. This review uses The Cancer Genome Atlas (TCGA) GBM dataset in combination with a data-mining approach to summarize disease characteristics, with a focus on select molecular pathways that drive GBM pathogenesis. We also present a unique genomic survey of RTKs that are frequently altered in GBM subtypes, as well as catalog the GBM disease association scores for all RTKs. Lastly, we discuss current RTK targeted therapies and highlight emerging directions in GBM research.

Adaptor Protein ShcD/SHC4 Interacts with Tie2 Receptor to Synergistically Promote Glioma Cell Invasion.

Gliomas are characterized by diffuse infiltration of tumor cells into surrounding brain tissue, and this highly invasive nature contributes to disease recurrence and poor patient outcomes. The molecular mechanisms underlying glioma cell invasion remain incompletely understood, limiting development of new targeted therapies. Here, we have identified phosphotyrosine adaptor protein ShcD as upregulated in malignant glioma and shown that it associates with receptor tyrosine kinase Tie2 to facilitate invasion. In human glioma cells, we find that expression of ShcD and Tie2 increases invasion, and this significant synergistic effect is disrupted with a ShcD mutant that cannot bind Tie2 or hyperphosphorylate the receptor. Expression of ShcD and/or Tie2 further increases invadopodia formation and matrix degradation in U87 glioma cells. In a coculture model, we show that U87-derived tumor spheroids expressing both ShcD and Tie2 display enhanced infiltration into cerebral organoids. Mechanistically, we identify changes in focal adhesion kinase phosphorylation in the presence of ShcD and/or Tie2 in U87 cells upon Tie2 activation. Finally, we identify a strong correlation between transcript levels of ShcD and Tie2 signaling components as well as N-cadherin in advanced gliomas and those with classical or mesenchymal subtypes, and we show that elevated expression of ShcD correlates with a significant reduction in patient survival in higher grade gliomas with mesenchymal signature. Altogether, our data highlight a novel Tie2-ShcD signaling axis in glioma cell invasion, which may be of clinical significance. IMPLICATIONS: ShcD cooperates with Tie2 to promote glioma cell invasion and its elevated expression correlates with poor patient outcome in advanced gliomas.

Multivalent nephrin-Nck interactions define a threshold for clustering and tyrosine-dependent nephrin endocytosis.

Assembly of signaling molecules into micrometer-sized clusters is driven by multivalent protein-protein interactions, such as those found within the nephrin-Nck (Nck1 or Nck2) complex. Phosphorylation on multiple tyrosine residues within the tail of the nephrin transmembrane receptor induces recruitment of the cytoplasmic adaptor protein Nck, which binds via its triple SH3 domains to various effectors, leading to actin assembly. The physiological consequences of nephrin clustering are not well understood. Here, we demonstrate that nephrin phosphorylation regulates the formation of membrane clusters in podocytes. We also reveal a connection between clustering and endocytosis, which appears to be driven by threshold levels of nephrin tyrosine phosphorylation and Nck SH3 domain signaling. Finally, we expose an in vivo correlation between transient changes in nephrin tyrosine phosphorylation, nephrin localization and integrity of the glomerular filtration barrier during podocyte injury. Altogether, our results suggest that nephrin phosphorylation determines the composition of effector proteins within clusters to dynamically regulate nephrin turnover and podocyte health.

Localization of phosphotyrosine adaptor protein ShcD/SHC4 in the adult rat central nervous system.

BACKGROUND: Mammalian Shc (Src homology and collagen) proteins comprise a family of four phosphotyrosine adaptor molecules which exhibit varied spatiotemporal expression and signaling functions. ShcD is the most recently discovered homologue and it is highly expressed in the developing central nervous system (CNS) and adult brain. Presently however, its localization within specific cell types of mature neural structures has yet to be characterized. RESULTS: In the current study, we examine the expression profile of ShcD in the adult rat CNS using immunohistochemistry, and compare with those of the neuronally enriched ShcB and ShcC proteins. ShcD shows relatively widespread distribution in the adult brain and spinal cord, with prominent levels of staining throughout the olfactory bulb, as well as in sub-structures of the cerebellum and hippocampus, including the subgranular zone. Co-localization studies confirm the expression of ShcD in mature neurons and progenitor cells. ShcD immunoreactivity is primarily localized to axons and somata, consistent with the function of ShcD as a cytoplasmic adaptor. Regional differences in expression are observed among neural Shc proteins, with ShcC predominating in the hippocampus, cerebellum, and some fiber tracts. Interestingly, ShcD is uniquely expressed in the olfactory nerve layer and in glomeruli of the main olfactory bulb. CONCLUSIONS: Together our findings suggest that ShcD may provide a distinct signaling contribution within the olfactory system, and that overlapping expression of ShcD with other Shc proteins may allow compensatory functions in the brain.

Signaling adaptor ShcD suppresses extracellular signal-regulated kinase (Erk) phosphorylation distal to the Ret and Trk neurotrophic receptors.

Proteins of the Src homology and collagen (Shc) family are typically involved in signal transduction events involving Ras/MAPK and PI3K/Akt pathways. In the nervous system, they function proximal to the neurotrophic factors that regulate cell survival, differentiation, and neuron-specific characteristics. The least characterized homolog, ShcD, is robustly expressed in the developing and mature nervous system, but its contributions to neural cell circuitry are largely uncharted. We now report that ShcD binds to active Ret, TrkA, and TrkB neurotrophic factor receptors predominantly via its phosphotyrosine-binding (PTB) domain. However, in contrast to the conventional Shc adaptors, ShcD suppresses distal phosphorylation of the Erk MAPK. Accordingly, genetic knock-out of mouse ShcD enhances Erk phosphorylation in the brain. In cultured cells, this capacity is tightly aligned to phosphorylation of ShcD CH1 region tyrosine motifs, which serve as docking platforms for signal transducers, such as Grb2. Erk suppression is relieved through independent mutagenesis of the PTB domain and the CH1 tyrosine residues, and successive substitution of these tyrosines breaks the interaction between ShcD and Grb2, thereby promoting TrkB-Grb2 association. Erk phosphorylation can also be restored in the presence of wild type ShcD through Grb2 overexpression. Conversely, mutation of the ShcD SH2 domain results in enhanced repression of Erk. Although the SH2 domain is a less common binding interface in Shc proteins, we demonstrate that it associates with the Ptpn11 (Shp2) phosphatase, which in turn regulates ShcD tyrosine phosphorylation. We therefore propose a model whereby ShcD competes with neurotrophic receptors for Grb2 binding and opposes activation of the MAPK cascade.

Nephrin Tyrosine Phosphorylation Is Required to Stabilize and Restore Podocyte Foot Process Architecture.

Podocytes are specialized epithelial cells of the kidney blood filtration barrier that contribute to permselectivity via a series of interdigitating actin-rich foot processes. Positioned between adjacent projections is a unique cell junction known as the slit diaphragm, which is physically connected to the actin cytoskeleton via the transmembrane protein nephrin. Evidence indicates that tyrosine phosphorylation of the intracellular tail of nephrin initiates signaling events, including recruitment of cytoplasmic adaptor proteins Nck1 and Nck2 that regulate actin cytoskeletal dynamics. Nephrin tyrosine phosphorylation is altered in human and experimental renal diseases characterized by pathologic foot process remodeling, prompting the hypothesis that phosphonephrin signaling directly influences podocyte morphology. To explore this possibility, we generated and analyzed knockin mice with mutations that disrupt nephrin tyrosine phosphorylation and Nck1/2 binding (nephrin(Y3F/Y3F) mice). Homozygous nephrin(Y3F/Y3F) mice developed progressive proteinuria accompanied by structural changes in the filtration barrier, including podocyte foot process effacement, irregular thickening of the glomerular basement membrane, and dilated capillary loops, with a similar but later onset phenotype in heterozygous animals. Furthermore, compared with wild-type mice, nephrin(Y3F/Y3F) mice displayed delayed recovery in podocyte injury models. Profiling of nephrin tyrosine phosphorylation dynamics in wild-type mice subjected to podocyte injury indicated site-specific differences in phosphorylation at baseline, injury, and recovery, which correlated with loss of nephrin-Nck1/2 association during foot process effacement. Our results define an essential requirement for nephrin tyrosine phosphorylation in stabilizing podocyte morphology and suggest a model in which dynamic changes in phosphotyrosine-based signaling confer plasticity to the podocyte actin cytoskeleton.

Advances in slit diaphragm signaling.

PURPOSE OF REVIEW: The podocyte slit diaphragm is a fundamental component of the glomerular filtration barrier and its function is highly dependent on the maintenance of specialized actin-based projections known as foot processes. In this review, we update the function of key slit diaphragm-associated proteins, and introduce some new players and emerging avenues of research within podocyte biology. RECENT FINDINGS: Studies using rodent models continue to support the long-held belief that precise regulation of actin dynamics at the slit diaphragm is essential for proper foot process organization. However, it is also becoming increasingly clear that alterations in actin remodeling can significantly contribute to damage in both animal models and human disease. In particular, the importance of signaling via the Rho family of GTPases has been recognized, as well as the requirement for proper localization and turnover of the slit diaphragm. SUMMARY: Regulation of the connection between the slit diaphragm and the podocyte actin network requires complex interplay between multiple signaling pathways. New discoveries contribute to an ever-expanding view of the slit diaphragm and serve to create a framework for the development of new therapeutic strategies targeting podocyte function in the future.

Direct regulation of nephrin tyrosine phosphorylation by Nck adaptor proteins.

The transmembrane protein nephrin is a key component of the kidney slit diaphragm that contributes to the morphology of podocyte foot processes through signaling to the underlying actin cytoskeleton. We have recently reported that tyrosine phosphorylation of the cytoplasmic tail of nephrin facilitates recruitment of Nck SH2/SH3 adaptor proteins and subsequent actin remodeling and that phosphorylation of the Nck binding sites on nephrin is decreased during podocyte injury. We now demonstrate that Nck directly modulates nephrin phosphorylation through formation of a signaling complex with the Src family kinase Fyn. The ability of Nck to enhance nephrin phosphorylation is compromised in the presence of a Src family kinase inhibitor and when the SH3 domains of Nck are mutated. Furthermore, induced loss of Nck expression in podocytes in vivo is associated with a rapid reduction in nephrin tyrosine phosphorylation. Our results suggest that Nck may facilitate dynamic signaling events at the slit diaphragm by promoting Fyn-dependent phosphorylation of nephrin, which may be important in the regulation of foot process morphology and response to podocyte injury.

Podocyte Protein, Nephrin, Is a Substrate of Protein Tyrosine Phosphatase 1B.

Glomerular podocytes are critical for the barrier function of the glomerulus in the kidney and their dysfunction causes protein leakage into the urine (proteinuria). Nephrin is a key podocyte protein, which regulates the actin cytoskeleton via tyrosine phosphorylation of its cytoplasmic domain. Here we report that two protein tyrosine phosphatases, PTP1B and PTP-PEST negatively regulate nephrin tyrosine phosphorylation. PTP1B directly binds to and dephosphorylates nephrin, while the action of PTP-PEST is indirect. The two phosphatases are also upregulated in the glomerulus in the rat model of puromycin aminonucleoside nephrosis. Both overexpression and inhibition of PTP1B deranged the actin cytoskeleton in cultured mouse podocytes. Thus, protein tyrosine phosphatases may affect podocyte function via regulating nephrin tyrosine phosphorylation.

Nck proteins maintain the adult glomerular filtration barrier.

Within the glomerulus, the scaffolding protein nephrin bridges the actin-rich foot processes that extend from adjacent podocytes to form the slit diaphragm. Mutations affecting a number of slit diaphragm proteins, including nephrin, cause glomerular disease through rearrangement of the actin cytoskeleton and disruption of the filtration barrier. We recently established that the Nck family of Src homology 2 (SH2)/SH3 cytoskeletal adaptor proteins can mediate nephrin-dependent actin reorganization. Formation of foot processes requires expression of Nck in developing podocytes, but it is unknown whether Nck maintains podocyte structure and function throughout life. Here, we used an inducible transgenic strategy to delete Nck expression in adult mouse podocytes and found that loss of Nck expression rapidly led to proteinuria, glomerulosclerosis, and altered morphology of foot processes. We also found that podocyte injury reduced phosphorylation of nephrin in adult kidneys. These data suggest that Nck is required to maintain adult podocytes and that phosphotyrosine-based interactions with nephrin may occur in foot processes of resting, mature podocytes.

Phosphorylated YDXV motifs and Nck SH2/SH3 adaptors act cooperatively to induce actin reorganization.

We have analyzed the means by which the Nck family of adaptor proteins couples adhesion proteins to actin reorganization. The nephrin adhesion protein is essential for the formation of actin-based foot processes in glomerular podocytes. The clustering of nephrin induces its tyrosine phosphorylation, Nck recruitment, and sustained localized actin polymerization. Any one of three phosphorylated (p)YDXV motifs on nephrin is sufficient to recruit Nck through its Src homology 2 (SH2) domain and induce localized actin polymerization at these clusters. Similarly, Nck SH3 mutants in which only the second or third SH3 domain is functional can mediate nephrin-induced actin polymerization. However, combining such nephrin and Nck mutants attenuates actin polymerization at nephrin-Nck clusters. We propose that the multiple Nck SH2-binding motifs on nephrin and the multiple SH3 domains of Nck act cooperatively to recruit the high local concentration of effectors at sites of nephrin activation that is required to initiate and maintain actin polymerization in vivo. We also find that YDXV motifs in the Tir protein of enteropathogenic Escherichia coli and nephrin are functionally interchangeable, indicating that Tir reorganizes the actin cytoskeleton by molecular mimicry of nephrin-like signaling. Together, these data identify pYDXV/Nck signaling as a potent and portable mechanism for physiological and pathological actin regulation.