Taking Snapshots of Ciliary Signaling

Although the primary cilium critically influences signaling in development and disease, the organelle’s small size and the dynamic nature of signaling events have posed obstacles to dissecting critical mechanisms. Reporting in Developmental Cell, Mick and colleagues (2015) describe cilia-APEX as an approach to provide dynamic “snapshots” of the ciliary proteome

Aurora A kinase activity influences calcium signaling in kidney cells

Aurora A is abnormally expressed and activated in cells lining cysts associated with polycystic kidney disease and can phosphorylate and inactivate polycystin 2

Ganetespib limits ciliation and cystogenesis in autosomal-dominant polycystic kidney disease (ADPKD)

© 2018 FASEB. Autosomal-dominant polycystic kidney disease (ADPKD) is associated with progressive formation of renal cysts, kidney enlargement, hypertension, and typically end-stage renal disease. In ADPKD, inherited mutations disrupt functionofthe polycystins (encoded byPKD1 and PKD2), thus causing loss of a cyst-repressive signal emanating from the renal cilium. Genetic studies have suggested ciliary maintenance is essential for ADPKD pathogenesis. Heat shock protein 90 (HSP90) clients include multiple proteins linked to ciliary maintenance. We determined that ganetespib, a clinical HSP90 inhibitor, inhibited proteasomal repression of NEK8 and the Aurora-A activator trichoplein, rapidly activating Aurora-A kinase and causing ciliary loss in vitro. Using conditional mouse models for ADPKD, we performed long-term (10 or 50 wk) dosing experiments that demonstrated HSP90 inhibition caused durable in vivo loss of cilia, controlled cystic growth, and ameliorated symptoms induced by loss of Pkd1 or Pkd2.Ganetespib efficacy was not increased by combination with 2-deoxy-D-glucose, aglycolysis inhibitor showing some promise for ADPKD. These studies identify a new biologic activity for HSP90 and support a cilia-based mechanism for cyst repression

A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system

BACKGROUND: Whether for cell culture studies of protein function, construction of mouse models to enable in vivo analysis of disease epidemiology, or ultimately gene therapy of human diseases, a critical enabling step is the ability to achieve finely controlled regulation of gene expression. Previous efforts to achieve this goal have explored inducible drug regulation of gene expression, and construction of synthetic promoters based on two-hybrid paradigms, among others. RESULTS: In this report, we describe the combination of dimerizer-regulated two-hybrid and tetracycline regulatory elements in an ordered cascade, placing expression of endpoint reporters under the control of two distinct drugs. In this Dual Drug Control (DDC) system, a first plasmid expresses fusion proteins to DBD and AD, which interact only in the presence of a small molecule dimerizer; a second plasmid encodes a cassette transcriptionally responsive to the first DBD, directing expression of the Tet-OFF protein; and a third plasmid encodes a reporter gene transcriptionally responsive to binding by Tet-OFF. We evaluate the dynamic range and specificity of this system in comparison to other available systems. CONCLUSION: This study demonstrates the feasibility of combining two discrete drug-regulated expression systems in a temporally sequential cascade, without loss of dynamic range of signal induction. The efficient layering of control levels allowed by this combination of elements provides the potential for the generation of complex control circuitry that may advance ability to regulate gene expression in vivo

Rapid calcium-dependent activation of Aurora-A kinase

Oncogenic hyperactivation of the mitotic kinase Aurora-A (AurA) in cancer is associated with genomic instability. Increasing evidence indicates that AurA also regulates critical processes in normal interphase cells, but the source of such activity has been obscure. We report here that multiple stimuli causing release of Ca2+ from intracellular endoplasmic reticulum stores rapidly and transiently activate AurA, without requirement for second messengers. This activation is mediated by direct Ca2+-dependent calmodulin (CaM) binding to multiple motifs on AurA. On the basis of structure–function analysis and molecular modelling, we map two primary regions of CaM-AurA interaction to unfolded sequences in the AurA N- and C-termini. This unexpected mechanism for AurA activation provides a new context for evaluating the function of AurA and its inhibitors in normal and cancerous cells

NEDD9 promotes oncogenic signaling, a stem/mesenchymal gene signature, and aggressive ovarian cancer growth in mice

© 2018, Macmillan Publishers Limited, part of Springer Nature. Neural precursor cell expressed, developmentally downregulated 9 (NEDD9) supports oncogenic signaling in a number of solid and hematologic tumors. Little is known about the role of NEDD9 in ovarian carcinoma (OC), but available data suggest elevated mRNA and protein expression in advanced stage high-grade cancers. We used a transgenic MISIIR-TAg mouse OC model combined with genetic ablation of Nedd9 to investigate its action in the development and progression of OC. A Nedd9−/− genotype delayed tumor growth rate, reduced incidence of ascites, and reduced expression and activation of signaling proteins including SRC, STAT3, E-cadherin, and AURKA. Cell lines established from MISIIR-TAg;Nedd9−/− and MISIIR-TAg;Nedd9+/+ mice exhibited altered migration and invasion. Growth of these cells in a syngeneic allograft model indicated that systemic Nedd9 loss in the microenvironment had little impact on tumor allograft growth, but in a Nedd9 wild-type background Nedd9−/− allografts exhibited significantly reduced growth, dissemination, and oncogenic signaling compared to Nedd9+/+ allografts. Gene expression analysis revealed that Nedd9+/+ tumors exhibited more mesenchymal “stem-like” transcriptional program, including increased expression of Aldh1a1 and Aldh1a2. Conversely, loss of Nedd9 resulted in increased expression of differentiation genes, including fallopian tube markers Foxj1, Ovgp1, and Pax8. Collectively, these data suggest that tumor cell-intrinsic Nedd9 expression promotes OC development and progression by broad induction of oncogenic protein signaling and stem/mesenchymal gene expression

Embryonal Fyn-associated substrate (EFS) and CASS4: The lesser-known CAS protein family members

© 2015 Elsevier B.V.. The CAS (Crk-associated substrate) adaptor protein family consists of four members: CASS1/BCAR1/p130Cas, CASS2/NEDD9/HEF1/Cas-L, CASS3/EFS/Sin and CASS4/HEPL. While CAS proteins lack enzymatic activity, they contain specific recognition and binding sites for assembly of larger signaling complexes that are essential for cell proliferation, survival, migration, and other processes. All family members are intermediates in integrin-dependent signaling pathways mediated at focal adhesions, and associate with FAK and SRC family kinases to activate downstream effectors regulating the actin cytoskeleton. Most studies of CAS proteins to date have been focused on the first two members, BCAR1 and NEDD9, with altered expression of these proteins now appreciated as influencing disease development and prognosis for cancer and other serious pathological conditions. For these family members, additional mechanisms of action have been defined in receptor tyrosine kinase (RTK) signaling, estrogen receptor signaling or cell cycle progression, involving discrete partner proteins such as SHC, NSP proteins, or AURKA. By contrast, EFS and CASS4 have been less studied, although structure-function analyses indicate they conserve many elements with the better-known family members. Intriguingly, a number of recent studies have implicated these proteins in immune system function, and the pathogenesis of developmental disorders, autoimmune disorders including Crohn's disease, Alzheimer's disease, cancer and other diseases. In this review, we summarize the current understanding of EFS and CASS4 protein function in the context of the larger CAS family group

Mechanisms for nonmitotic activation of Aurora-A at cilia

© 2017 The Author(s).Overexpression of the Aurora kinase A (AURKA) is oncogenic in many tumors. Many studies of AURKA have focused on activities of this kinase in mitosis, and elucidated the mechanisms by which AURKA activity is induced at the G2/M boundary through interactions with proteins such as TPX2 and NEDD9. These studies have informed the development of small molecule inhibitors of AURKA, of which a number are currently under preclinical and clinical assessment. While the first activities defined for AURKA were its control of centrosomal maturation and organization of the mitotic spindle, an increasing number of studies over the past decade have recognized a separate biological function of AURKA, in controlling disassembly of the primary cilium, a small organelle protruding from the cell surface that serves as a signaling platform. Importantly, these activities require activation of AURKA in early G1, and the mechanisms of activation are much less well defined than those in mitosis. A better understanding of the control of AURKA activity and the role of AURKA at cilia are both important in optimizing the efficacy and interpreting potential downstream consequences of AURKA inhibitors in the clinic. We here provide a current overview of proteins and mechanisms that have been defined as activating AURKA in G1, based on the study of ciliary disassembly

Analysis of the application of the Black- Scholes model on the FTSE/ASE-20 stock options of the Athens Derivatives Exchange

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