Phosphorylation Of Domain Research Articles

Structure and activation mechanism of the hexameric plasma membrane H+-ATPase

The S. cerevisiae plasma membrane H+-ATPase, Pma1, is a P3A-type ATPase and the primary protein component of the membrane compartment of Pma1 (MCP). Like other plasma membrane H+-ATPases, Pma1 assembles and functions as a hexamer, a property unique to this subfamily among the larger family of P-type ATPases. It has been unclear how Pma1 organizes the yeast membrane into MCP microdomains, or why it is that Pma1 needs to assemble into a hexamer to establish the membrane electrochemical proton gradient. Here we report a high-resolution cryo-EM study of native Pma1 hexamers embedded in endogenous lipids. Remarkably, we found that the Pma1 hexamer encircles a liquid-crystalline membrane domain composed of 57 ordered lipid molecules. The Pma1-encircled lipid patch structure likely serves as the building block of the MCP. At pH 7.4, the carboxyl-terminal regulatory α-helix binds to the phosphorylation domains of two neighboring Pma1 subunits, locking the hexamer in the autoinhibited state. The regulatory helix becomes disordered at lower pH, leading to activation of the Pma1 hexamer. The activation process is accompanied by a 6.7 Å downward shift and a 40° rotation of transmembrane helices 1 and 2 that line the proton translocation path. The conformational changes have enabled us to propose a detailed mechanism for ATP-hydrolysis-driven proton pumping across the plasma membrane. Our structures will facilitate the development of antifungal drugs that target this essential protein.

Activation of the STAT1-BCL-2/MCL-1 Axis in Leukemic Cells Carrying a SPAG9-JAK2 Fusion

[Background and aim of this study]Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a distinct subtype of B-ALL with poor prognosis. JAK2 related fusion genes have been identified in this subtype, especially in adolescent and young adults (AYA). Previously, we identified SPAG9-JAK2 fusion gene in 14-year-old boy with Ph-like ALL (Kawamura M, et al. Genes Chromosomes Cancer. 2015). In this study, we performed functional analysis of the SPAG9-JAK2 fusion protein, and evaluated the efficacy of treatment with a JAK inhibitor against cells carrying the fusion. In addition, we assessed therapeutic options other than JAK2 inhibition following comprehensive molecular analysis.[Materials and Methods]Full length of SPAG9-JAK2 cDNA was cloned into retroviral construct with Tet-On system. Ba/F3 cells, which are IL-3 dependent murine pro B-ALL cells, were transduced with retroviral vector to establish Ba/F3 cells expressing SPAG9-JAK2 (Ba/F3-SPAG9-JAK2) under doxycycline (DOX) dependent manner. Ba/F3-SPAG9-JAK2 were analyzed whether IL-3 independent growth was achieved. Aberrant activation of JAK-STAT pathway achieved by SPAG9-JAK2 was evaluated by western blot. To clarify whether the tyrosine residues of JAK2 in this fusion protein were critical for IL-3-independent proliferation, Ba/F3 cells expressing SPAG9-JAK2 mutants (SPAG9-JAK2 mut) in which both tyrosine residues of JAK2 were replaced with phenylalanine was established. Gene expression analysis using Mouse Genome 430 2.0 Array was performed for comprehensive analysis of gene expression profile related to SPAG9-JAK2. Sensitivity of Ba/F3-SPAG9-JAK2 to ruxolitinib (a JAK inhibitor) was tested in cytotoxic assay and in xenograft model established using Ba/F3-SPAG9-JAK2 cells.[Results and discussions]The expression of SPAG9-JAK2 in Ba/F3 cells under DOX dependent manner was confirmed by western blot. Ba/F3-SPAG9-JAK2 proliferated without IL-3 in contrast to Ba/F3 cells (p<0.01), suggesting SPAG9-JAK2 had the proliferation activity. Western blot revealed that constitutive phosphorylation of tyrosine residue of SPAG9-JAK2, STAT3/STAT5, suggesting constitutive activation of JAK2-STAT3/STAT5 pathway. SPAG9-JAK2 mut abolished IL-3 independence (p<0.01), but had no influence on STAT3/STAT5 phosphorylation levels detected by western blot. Gene expression analysis revealed that Stat1 was significantly up-regulated in Ba/F3-SPAG9-JAK2 cells compared with mock Ba/F3 cells (fold change 8.04 with p < 0.01) [Fig. 1], confirmed by western blot. STAT1 was also phosphorylated in Ba/F3-SPAG9-JAK2 but not SPAG9-JAK2 mut cells detected by western blot [Fig. 2], suggesting that STAT1 is a key mediator for SPAG9-JAK2-mediated cell proliferation. Consistently, STAT1 induced expression of the anti-apoptotic proteins, BCL-2 and MCL-1, as did SPAG9-JAK2, but not SPAG9-JAK2 mut confirmed by western blot [Fig. 3]. Ruxolitinib abrogated Ba/F3-SPAG9-JAK2-mediated proliferation in vitro (p<0.01), with an 50% inhibitory concentration (IC50) value of 65.9 ± 9.8 nM, causing decrease of JAK2, STAT1/STAT3/STAT5 phospholyration in western blot and apoptosis in annexin V/PI staining. Ruxolitinib prolonged survival time of xenotransplanted mice (p = 0.0213), however, the proliferation of leukemic cells in mouse bone marrow was not suppressed by ruxolitinib. Ba/F3-SPAG9-JAK2 cells showed a dose-dependent response for venetoclax (a BCL-2 inhibitor) with IC50 2.57 ± 1.11 µM and AZD5991 (an MCL-1 inhibitor) with IC50 6.76 ± 3.31 µM. Treatment of Ba/F3-SPAG9-JAK2 cells with a combination of ruxolitinib and venetoclax or AZD5991 resulted in a significant reduction in the IC50 of ruxolitinib (p<0.01) [Fig. 4, 5], with a combined index (CI) value of 0.61 or 0.92, indicating a moderately or weak synergistic effect in vitro.[Conclusion]SPAG9-JAK2 promotes cell proliferation and that tyrosine phosphorylation of the JAK2 kinase domain is critical for IL-3-independent cell growth. Ruxolitinib shows sufficient cytotoxic effects against Ba/F3-SPAG9-JAK2 cells in vitro, but is only partially effective in vivo. Activation of the JAK2-STAT1-BCL-2/MCL-1 axis contributes to aberrant growth promotion by SPAG9-JAK2. BCL-2 or MCL-1 inhibitors in combination with ruxolitinib shows efficacy against Ph-like ALL carrying the SPAG9-JAK2 fusion in vitro. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Regulation of CYLD activity and specificity by phosphorylation and ubiquitin-binding CAP-Gly domains.

SummaryNon-degradative ubiquitin chains and phosphorylation events govern signaling responses by innate immune receptors. The deubiquitinase CYLD in complex with SPATA2 is recruited to receptor signaling complexes by the ubiquitin ligase LUBAC and regulates Met1- and Lys63-linked polyubiquitin and receptor signaling outcomes. Here, we investigate the molecular determinants of CYLD activity. We reveal that two CAP-Gly domains in CYLD are ubiquitin-binding domains and demonstrate a requirement of CAP-Gly3 for CYLD activity and regulation of immune receptor signaling. Moreover, we identify a phosphorylation switch outside of the catalytic USP domain, which activates CYLD toward Lys63-linked polyubiquitin. The phosphorylated residue Ser568 is a novel tumor necrosis factor (TNF)-regulated phosphorylation site in CYLD and works in concert with Ser418 to enable CYLD-mediated deubiquitination and immune receptor signaling. We propose that phosphorylated CYLD, together with SPATA2 and LUBAC, functions as a ubiquitin-editing complex that balances Lys63- and Met1-linked polyubiquitin at receptor signaling complexes to promote LUBAC signaling.

3D Domain Swapping Dimerization of the Receiver Domain of Cytokinin Receptor CRE1 From Arabidopsis thaliana and Medicago truncatula.

Cytokinins are phytohormones regulating many biological processes that are vital to plants. CYTOKININ RESPONSE1 (CRE1), the main cytokinin receptor, has a modular architecture composed of a cytokinin-binding CHASE (Cyclases/Histidine kinases Associated Sensory Extracellular) domain, followed by a transmembrane fragment, an intracellular histidine kinase (HK) domain, and a receiver domain (REC). Perception of cytokinin signaling involves (i) a hormone molecule binding to the CHASE domain, (ii) CRE1 autophosphorylation at a conserved His residue in the HK domain, followed by a phosphorelay to (iii) a conserved Asp residue in the REC domain, (iv) a histidine-containing phosphotransfer protein (HPt), and (v) a response regulator (RR). This work focuses on the crystal structures of the REC domain of CRE1 from the model plant Arabidopsis thaliana and from the model legume Medicago truncatula. Both REC domains form tight 3D-domain-swapped dimers. Dimerization of the REC domain agrees with the quaternary assembly of the entire CRE1 but is incompatible with a model of its complex with HPt, suggesting that a considerable conformational change should occur to enable the signal transduction. Indeed, phosphorylation of the REC domain can change the HPt-binding properties of CRE1, as shown by functional studies.

Phosphorylated cingulin localises GEF-H1 at tight junctions to protect vascular barriers in blood endothelial cells.

ABSTRACTDysfunction of vascular barriers is a critical step in inflammatory diseases. Endothelial tight junctions (TJs) control barrier function, and the cytoplasmic adaptor protein cingulin connects TJs to signalling pathways. However, local events at TJs during inflammation are largely unknown. In this study, we investigate the local response of TJ adaptor protein cingulin and its interaction with Rho guanine nucleotide exchange factor H1 (GEF-H1, also known as ARHGEF2) upon vascular barrier disruption to find a new approach to counteract vascular leak. Based on transendothelial-electrical-resistance (TEER) measurements, cingulin strengthened barrier integrity upon stimulation with histamine, thrombin and VEGF. Cingulin also attenuated myosin light chain 2 (MLC2; also known as MYL2) phosphorylation by localising GEF-H1 to cell junctions. By using cingulin phosphomutants, we verified that the phosphorylation of the cingulin head domain is required for its protective effect. Increased colocalisation of GEF-H1 and cingulin was observed in the vessels of vasculitis patients compared to those in healthy skin. Our findings demonstrate that cingulin can counteract vascular leak at TJs, suggesting the existence of a novel mechanism in blood endothelial cells that protects barrier function during disease.

Structural basis for ALK2/BMPR2 receptor complex signaling through kinase domain oligomerization

Upon ligand binding, bone morphogenetic protein (BMP) receptors form active tetrameric complexes, comprised of two type I and two type II receptors, which then transmit signals to SMAD proteins. The link between receptor tetramerization and the mechanism of kinase activation, however, has not been elucidated. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, combined with analysis of SMAD signaling, we show that the kinase domain of the type I receptor ALK2 and type II receptor BMPR2 form a heterodimeric complex via their C-terminal lobes. Formation of this dimer is essential for ligand-induced receptor signaling and is targeted by mutations in BMPR2 in patients with pulmonary arterial hypertension (PAH). We further show that the type I/type II kinase domain heterodimer serves as the scaffold for assembly of the active tetrameric receptor complexes to enable phosphorylation of the GS domain and activation of SMADs.

Radiation-Induced Phosphorylation of a Prion-Like Domain Regulates Transformation by FUS-CHOP.

Chromosomal translocations generate oncogenic fusion proteins in approximately one-third of sarcomas, but how these proteins promote tumorigenesis is not well understood. Interestingly, some translocation-driven cancers exhibit dramatic clinical responses to therapy, such as radiotherapy, although the precise mechanism has not been elucidated. Here we reveal a molecular mechanism by which the fusion oncoprotein FUS-CHOP promotes tumor maintenance that also explains the remarkable sensitivity of myxoid liposarcomas to radiation therapy. FUS-CHOP interacted with chromatin remodeling complexes to regulate sarcoma cell proliferation. One of these chromatin remodelers, SNF2H, colocalized with FUS-CHOP genome-wide at active enhancers. Following ionizing radiation, DNA damage response kinases phosphorylated the prion-like domain of FUS-CHOP to impede these protein-protein interactions, which are required for transformation. Therefore, the DNA damage response after irradiation disrupted oncogenic targeting of chromatin remodelers required for FUS-CHOP-driven sarcomagenesis. This mechanism of disruption links phosphorylation of the prion-like domain of an oncogenic fusion protein to DNA damage after ionizing radiation and reveals that a dependence on oncogenic chromatin remodeling underlies sensitivity to radiation therapy in myxoid liposarcoma. SIGNIFICANCE: Prion-like domains, which are frequently translocated in cancers as oncogenic fusion proteins that drive global epigenetic changes, confer sensitivity to radiation via disruption of oncogenic interactions.

Pharmaceutical Interference of the EWS-FLI1-driven Transcriptome By Cotargeting H3K27ac and RNA Polymerase Activity in Ewing Sarcoma.

The EWSR1-FLI1 t(11;22)(q24;q12) translocation is the hallmark genomic alteration of Ewing sarcoma, a malignancy of the bone and surrounding tissue, predominantly affecting children and adolescents. Although significant progress has been made for the treatment of localized disease, patients with metastasis or who relapse after chemotherapy have less than a 30% five-year survival rate. EWS-FLI1 is currently not clinically druggable, driving the need for more effective targeted therapies. Treatment with the H3K27 demethylase inhibitor, GSK-J4, leads to an increase in H3K27me and a decrease in H3K27ac, a significant event in Ewing sarcoma because H3K27ac associates strongly with EWS-FLI1 binding at enhancers and promoters and subsequent activity of EWS-FLI1 target genes. We were able to identify targets of EWS-FLI1 tumorigenesis directly inhibited by GSK-J4. GSK-J4 disruption of EWS-FLI1-driven transcription was toxic to Ewing sarcoma cells and slowed tumor growth in patient-derived xenografts (PDX) of Ewing sarcoma. Responses were markedly exacerbated by cotreatment with a disruptor of RNA polymerase II activity, the CDK7 inhibitor THZ1. This combination together suppressed EWS-FLI1 target genes and viability of ex vivo PDX Ewing sarcoma cells in a synergistic manner. In PDX models of Ewing Sarcoma, the combination shrank tumors. We present a new therapeutic strategy to treat Ewing sarcoma by decreasing H3K27ac at EWS-FLI1-driven transcripts, exacerbated by blocking phosphorylation of the C-terminal domain of RNA polymerase II to further hinder the EWS-FLI1-driven transcriptome.

MX2-mediated innate immunity against HIV-1 is regulated by serine phosphorylation.

The antiviral cytokine interferon activates expression of interferon-stimulated genes to establish an antiviral state. Myxovirus resistance 2 (MX2, also known as MxB) is an interferon-stimulated gene that inhibits the nuclear import of HIV-1 and interacts with the viral capsid and cellular nuclear transport machinery. Here, we identified the myosin light chain phosphatase (MLCP) subunits myosin phosphatase target subunit 1 (MYPT1) and protein phosphatase 1 catalytic subunit-β (PPP1CB) as positively-acting regulators of MX2, interacting with its amino-terminal domain. We demonstrated that serine phosphorylation of the N-terminal domain at positions 14, 17 and 18 suppresses MX2 antiviral function, prevents interactions with the HIV-1 capsid and nuclear transport factors, and is reversed by MLCP. Notably, serine phosphorylation of the N-terminal domain also impedes MX2-mediated inhibition of nuclear import of cellular karyophilic cargo. We also found that interferon treatment reduces levels of phosphorylation at these serine residues and outline a homeostatic regulatory mechanism in which repression of MX2 by phosphorylation, together with MLCP-mediated dephosphorylation, balances the deleterious effects of MX2 on normal cell function with innate immunity against HIV-1.

Discovery of a novel CDK7 inhibitor YPN-005 in small cell lung cancer

In contrast to non-small cell lung cancer, there has been no significant progress in the development of therapies for the small cell lung cancer (SCLC) in recent decades. Although various targeted agents, including immunotherapies, have recently been developed for testing in clinical trials, novel therapeutic agents are still needed for SCLC. We developed a potent inhibitor of cyclin-dependent kinase 7 (CDK7), designated YPN-005, and sought to determine whether it showed any anticancer effects in SCLC cells, cisplatin or etoposide-resistant cells, or organoids derived from SCLC patients. In a panel of kinases assay, YPN-005 was highly selective for CDK7 and showed antiproliferative effects in SCLC and cells with acquired resistance to conventional anticancer drugs. Similar to other CDK7 inhibitors, YPN-005 treatment significantly decreased the phosphorylation of the carboxyl-terminal domain of RNA polymerase II. Consistent with the in vitro results, YPN-005 treatment showed a significant inhibition of tumor growth through the suppression of RNA polymerase II phosphorylation. Finally, YPN-005 showed potent anticancer effects in organoids derived from SCLC patients compared to another CDK7 inhibitor, THZ1. Therapeutic targeting of CDK7 in SCLC might be suitable for clinical investigation, and YPN-005 may be a promising therapeutic option for primary SCLC and SCLC with acquired resistance to conventional therapy.

Canonical Wnt Pathway Is Activated in Myeloid Leukemia Stem Cells Via Hematopoietic Cell Kinase That Is Phosphorylated By an Autocrine TIM-3/Galectin-9 Signaling

(Introduction)Activation of β-catenin is critical for self-renewal of cancer stem cells (CSCs). A number of recurrent mutations related to the aberrant accumulation and activation of β-catenin have been identified in CSCs in various types of cancer, but such somatic mutations are barely found in leukemic stem cells (LSCs) in acute myeloid leukemia (AML). We have recently identified the autocrine loop consisted of LSC-specific surface molecule T-cell immunoglobulin mucin-3 (TIM-3) and its ligand galectin-9 (Gal-9) in human myeloid malignancies. When Gal-9 binds to TIM-3, TIM-3 signaling induces accumulation of β-catenin in nucleus (Kikushige et al, Cell Stem Cell, 2015). In this study, we sought to identify the molecular mechanism of aberrant β-catenin activation in LSCs of AML.(Results)To test whether TIM-3 signaling can activate the canonical Wnt pathway, we evaluated the activation of LDL receptor-related protein 6 (LRP6), a key component of canonical Wnt pathway. Immunoprecipitation assay by using anti-LRP6 antibody revealed that Gal-9 stimulation promoted the formation of the “LRP6-signalosome”, the protein complex consisted of Wnt components including LRP6, Axin and Dvl. Gal-9 stimulation also induced phosphorylation of LRP6at several sites including Thr1479 and subsequent β-catenin accumulation in primary TIM-3+ AML cells. On the other hand, this effect on LRP6 induced by Gal-9 stimulation was completely canceled in the presence of an anti-TIM-3 antibody (F38-2E2), which can block the TIM-3/Gal-9 interaction. Since the Wnt ligand is not involved in this assay system, the TIM-3/Gal-9 interaction should directly activate the canonical Wnt pathway, not through the previously identified Wnt signaling machinery.Gal-9 stimulation induced phosphorylation of cytoplasmic tail of TIM-3, which induces recruitment of several Src family kinases (SFKs). We found that Gal-9 ligation to TIM-3 activated several SFKs including hematopoietic cell kinase (HCK) by phosphorylation of the SH1 domain (Tyr410 in HCK) in TIM-3+ AML cells. Furthermore, an inhibitor specific for HCK, but not inhibitors effective for other SFKs, inhibited LRP6 phosphorylation induced by TIM-3/Gal-9 signaling, indicating that HCK should be a critical mediator for TIM-3 signaling in human LSCs.Next, we focused on p120-catenin because this molecule is known as not only a substrate for SFKs but also an indispensable molecule for the initial step of the LRP6-signalosome formation. We found that the phosphorylation of p120-catenin at Tyr228 was induced in response to Gal-9 stimulation. HCK inhibitors efficiently blocked the Tyr228 phosphorylation and subsequent activation of canonical β-catenin pathway. These results suggested that HCK activation induced by TIM-3/Gal-9 ligation could activate canonical β-catenin pathway through phosphorylation of p120-catenin.(Conclusion)In myeloid leukemia, TIM-3/Gal-9 signaling activates HCK, which induces p120-catenin phosphorylation, eventually acitavate the canonical β-catenin pathway, and these processes should be independent of Wnt ligands. Since TIM-3/Gal-9 autocrine loop signaling is constitutively active in AML, this novel machinery should contribute to the maintenance of AML LSCs through accumulation of β-catenin. Our study suggest that TIM-3 and Gal-9 as well as HCK should be important therapeutic targets in AML LCSs. DisclosuresNo relevant conflicts of interest to declare.

CDK9 Inhibition with LDC526 Is an Effective Treatment for T-Cell Prolymphocytic Leukemia (T-PLL) in Vitro

▪IntroductionT-cell prolymphocytic leukemia (T-PLL) is a rare leukemia with an aggressive disease course, characterized by chemotherapy resistance and a median overall survival of less than two years. Apart from defects in DNA damage response genes like ATM and genes of JAK/STAT signaling, overexpression of MYC, often caused by gain of chromosomal material, is frequently observed in T-PLL. Intravenous application of the anti-CD52 antibody Alemtuzumab has been demonstrated to improve progression-free survival and is currently considered the standard of care. In younger patients remissions can be consolidated by allogeneic stem cell transplantation, which results in long-term remission in a small subgroup of patients. The prognosis in the majority of patients relapsing from their disease is dismal and thus novel treatment options are urgently needed. Cyclin-dependent kinase 9 (CDK9), a serine/ threonine kinase which is ubiquitously expressed, is the catalytic unit of the positive transcription elongation factor (P-TEFb). In the presence of cyclin T, p-TEFb stimulates transcription elongation by phosphorylation of the carboxy-terminal domain (CTD) of RNA polymerase II (RNA Pol II). CDK9 inhibition by LDC526, a specific CDK9 inhibitor, leads to reduced phosphorylation of CTD-Ser2, Ser5 and Ser7 of RNA Pol II and subsequently to the downregulation of MYC and MCL-1.Methods</spanCD3+CD4+ tumor cells from cryopreserved peripheral blood samples of 11 T-PLL patients were enriched to a purity of >95% by fluorescence-activated cell sorting (FACS) or magnetic activated cell separation (MACS). RNA and DNA were isolated using the RNeasy Mini Kit® or the QIAamp DNA blood midi kit® (Qiagen, Hilden, Germany), respectively. To determine IC50 values, we performed cell viability assays for T-PLL cells of 11 patients treated with LDC526 and a panel of reference compounds compared to DMSO controls (CellTiter-Glo Luminescent Cell Viability Assay; Promega, Fitchburg, WI, USA). Gene expression profiling was performed in primary T-PLL cells treated with the CDK9 inhibitor LDC526 in a concentration of 10µM compared to DMSO (n=3) on Clariom S Pico Assays (Affymetrix, Santa Clara, CA, USA) and analyzed by gene set enrichment analysis (GSEA) and GeneTrail analysis. Gene expression measurement was performed with quantitative real-time PCR (qPCR) for MYC (Taqman, Thermo Fisher Scientific, Waltham, MA, USA). Protein expression was analyzed for pCTD Ser2, Ser5 and Ser7, MYC, p-STAT3 and MCL-1 by Western Blot (n=4) after LDC526-treatment in three different concentrations (10, 30 and 100 µM).ResultsLDC526 inhibited T-PLL cell survival in vitro in a dose-dependent manner. The median IC50 was 1.7 µM (range: 0.9 -3.5) which compared favourably to a panel of reference compounds including fludarabine, clofarabine, methotrexate, cytarabine and ruxolitinib (median IC50 values 6.4, 6.2, 29.9, 8.3, and 29.2, respectively). Similar experiments with BAY1143572, a structurally closely related CDK9 inhibitor, which is currently investigated in phase I studies of acute myeloid leukemia, revealed an even lower median IC50 of 0.9 µM (range: 0.5 - 1.7). Of note, the purine analogues fludarabine and clofarabine were only active in some of the patients tested, whereas both CDK9 inhibitors showed activity in all samples analyzed.Gene expression profiles of LDC526 treated T-PLL cells compared to DMSO controls revealed an enrichment of genes in gene sets associated with interferon and TNFA signatures, MYC targets as well as JAK/STAT genes, which were subsequently confirmed by qPCR experiments. At the protein level LDC526 dose-dependently reduced phosphorylation of CTD-Ser2, Ser7 and to a lesser extent of Ser5 compared to DMSO controls resulting in efficient down-regulation of MYC, MCL-1 and p-STAT3.ConclusionsOur experiments identify CDK9 as a novel therapeutic target in T-PLL which can be efficiently inhibited by LDC526 and BAY1143572 at clinically meaningful concentrations. CDK9 inhibition resulted in both reduced T-PLL cell survival and down-regulation of MYC and JAK/STAT signaling, which have a key role in the molecular pathogenesis of T-PLL. DisclosuresDürig:Lead Discovery Center: Research Funding.

Interhelical H-Bonds Modulate the Activity of a Polytopic Transmembrane Kinase.

DesK is a Histidine Kinase that allows Bacillus subtilis to maintain lipid homeostasis in response to changes in the environment. It is located in the membrane, and has five transmembrane helices and a cytoplasmic catalytic domain. The transmembrane region triggers the phosphorylation of the catalytic domain as soon as the membrane lipids rigidify. In this research, we study how transmembrane inter-helical interactions contribute to signal transmission; we designed a co-expression system that allows studying in vivo interactions between transmembrane helices. By Alanine-replacements, we identified a group of polar uncharged residues, whose side chains contain hydrogen-bond donors or acceptors, which are required for the interaction with other DesK transmembrane helices; a particular array of H-bond- residues plays a key role in signaling, transmitting information detected at the membrane level into the cell to finally trigger an adaptive response.

Multiple Blockades of the HGF/Met Signaling Pathway for Metastasis Suppression Using Nanoinhibitors.

The hepatocyte growth factor (HGF)/HGF receptor (Met) signaling pathway serves as a potential target for preventing tumor metastasis yet poorly explored. Here, we developed a Met-targeted nanoinhibitor to efficiently suppress metastasis via a multiple blockading HGF/Met signaling pathway. A biocompatible nanovector comprising multiple type of inhibitors enables interrupting extracellular domain dimerization and intracellular domain phosphorylation simultaneously. Such a comprehensive blockade of signaling pathway restrains unregulated tumor cell migration, invasion, and proliferation and thus remarkably suppresses metastasis in an orthotopic breast tumor model. This method provides a safe and effective option for metastasis inhibition via modulation of the cell signaling pathway. To our best knowledge, the strategy of the multiple blockading signaling pathway has not been reported for preventing tumor metastasis.

Abstract P-18: Phosphorylation of RNA Polymerase II C-Terminal Domain Affects Transcript Elongation Through Chromatin

Background: Transcription is the central point of gene regulation where the efficient maintenance of chromatin structure during the passage of RNA polymerase (Pol II) is critical for cell survival and functioning. The phosphorylation of carboxy-terminal domain (CTD) of the large subunit (Rpb1) of Pol II plays a key role in transcription through chromatin providing the binding and dissociation of factors essential for the mRNA biogenesis. Although the regulatory effect of chromatin structure on multiple stages of transcription has been well established, the role of CTD phosphorylation itself has not been systematically addressed. Methods: The effect of differentially phosphorylated Pol II-CTD on transcript elongation through chromatin was studied using in vitro transcription system based on mononucleosomes precisely positioned on DNA. The unphosphorylated and hyperphosphorylated Pol II-CTD were obtained using yeast genetics as well as in vitro kinase or phosphatases. Transcription rate and positions of pausing were measured using authentic elongation complexes comprising Pol II having different CTD phosphorylation states. The quantitative analysis of the transcripts was conducted using denaturing PAGE. Results: We observed a significant difference in the transcription through chromatin depending on CTD phosphorylation level. Thus, experiments on transcription of nucleosomes with Pol II isoforms have shown that the hyperphosphorylated form more efficiently transcribes the nucleosome and leads to a faster accumulation of the full-length RNA product than the non-phosphorylated isoform of Pol II. The non-phosphorylated isoform of the enzyme is characterized by a stronger pause in the early nucleosomal region and a slower accumulation of the full-length RNA product. Conclusion: Hyperphosphorylated form more efficiently transcribes the nucleosome and leads to a faster accumulation of the full-length RNA product as compared with the non-phosphorylated isoform of Pol II. A preliminary model of the effect of Pol II hyperphosphorylation on nucleosomal DNA transcription is proposed.

The Spf1p P5A-ATPase “arm-like” domain is not essential for ATP hydrolysis but its deletion impairs autophosphorylation

The yeast Spf1p P5A-ATPase actively translocates membrane spanning peptides of mislocalized proteins from the endoplasmic reticulum. Loss of Spf1p function causes a pleiotropic ER stress-phenotype associated with alterations of homeostasis of metal ions, lipids, protein folding, glycosylation, and membrane insertion. A unique characteristic of P5A-ATPases is the presence of an extended insertion which was called the “arm-like” domain connecting the phosphorylation domain (P) with transmembrane segment M5 near the peptidyl-substrate binding pocket. Here we have constructed and characterized a Δarm mutant of Spf1p lacking a segment of 117 amino acids of the “arm-like” domain. The Δarm mutant was capable of hydrolyzing ATP at maximal rates of 50% of that of the wild type enzyme. With the non-nucleotide substrate analog pNPP, the hydrolytic activity of the mutant dropped to 10%. The mutant showed an apparent affinity for ATP similar to the wild type. When incubated with ATP the Δarm mutant produced a lower level of the catalytic phosphoenzyme in amounts proportionate to the ATPase activity. These results indicate that the “arm-like” domain is not essential for hydrolytic activity and suggest that it is needed for the stabilization of Spf1p in a phosphorylation-ready conformation.

The Chlamydia trachomatis Tarp effector targets the Hippo pathway

Chlamydia trachomatis injects bacterial effector proteins into human epithelial cells to facilitate the establishment of new infections. The chlamydial type III secreted effector translocated actin recruiting phosphoprotein (Tarp) has been shown to nucleate and bundle actin filaments. It is also believed to initiate new signaling pathways via an N-terminal phosphorylation domain. A comprehensive understanding of the host pathways that are controlled by Tarp to aid in the establishment of a successful infection remains incomplete. To gain further insight into the cell signaling regulated by Tarp, we generated transgenic fruit flies engineered to express the N-terminal domain of Tarp. As many signaling pathways are conserved between flies and mammals, we hypothesized that expression of the Tarp N-domain in the fruit fly might disrupt key pathways, resulting in developmental defects. Tarp N-domain expression in the fruit fly resulted in a mechanosensory bristle duplication phenotype similar to a previously characterized fly phenotype found to be a consequence of defects in the Hippo pathway. Tarp-dependent disruption of the Hippo pathway was confirmed in a C. trachomatis tissue culture infection model. The capability of Tarp to alter Hippo pathway signaling in infected epithelial cells is a previously unrecognized pathway commandeered by chlamydia and likely contributes to the establishment of chlamydia's intracellular niche.

Structural basis for allosteric control of the SERCA-Phospholamban membrane complex by Ca2+ and phosphorylation.

Phospholamban (PLN) is a mini-membrane protein that directly controls the cardiac Ca2+-transport response to β-adrenergic stimulation, thus modulating cardiac output during the fight-or-flight response. In the sarcoplasmic reticulum membrane, PLN binds to the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), keeping this enzyme's function within a narrow physiological window. PLN phosphorylation by cAMP-dependent protein kinase A or increase in Ca2+ concentration reverses the inhibitory effects through an unknown mechanism. Using oriented-sample solid-state NMR spectroscopy and replica-averaged NMR-restrained structural refinement, we reveal that phosphorylation of PLN's cytoplasmic regulatory domain signals the disruption of several inhibitory contacts at the transmembrane binding interface of the SERCA-PLN complex that are propagated to the enzyme's active site, augmenting Ca2+ transport. Our findings address long-standing questions about SERCA regulation, epitomizing a signal transduction mechanism operated by posttranslationally modified bitopic membrane proteins.

Phosphorylation of the WH2 domain in yeast Las17/WASP regulates G-actin binding and protein function during endocytosis

Actin nucleation is the key rate limiting step in the process of actin polymerization, and tight regulation of this process is critical to ensure actin filaments form only at specific times and at defined regions of the cell. WH2 domains are short sequence motifs found in many different actin binding proteins including WASP family proteins which regulate the actin nucleating complex Arp2/3. In this study we reveal a phosphorylation site, Serine 554, within the WH2 domain of the yeast WASP homologue Las17. Both phosphorylation and a phospho-mimetic mutation reduce actin monomer binding affinity while an alanine mutation, generated to mimic the non-phosphorylated state, increases actin binding affinity. The effect of these mutations on the Las17-dependent process of endocytosis in vivo was analysed and leads us to propose that switching of Las17 phosphorylation states may allow progression through distinct phases of endocytosis from site assembly through to the final scission stage. While the study is focused on Las17, the sole WASP family protein in yeast, our results have broad implications for our understanding of how a key residue in this conserved motif can underpin the many different actin regulatory roles with which WH2 domains have been associated.

Toll receptors remodel epithelia by directing planar-polarized Src and PI3K activity

Toll-like receptors are essential for animal development and survival, with conserved roles in innate immunity, tissue patterning, and cell behavior. The mechanisms by which Toll receptors signal to the nucleus are well characterized, but how Toll receptors generate rapid, localized signals at the cell membrane to produce acute changes in cell polarity and behavior is not known. We show that Drosophila Toll receptors direct epithelial convergent extension by inducing planar-polarized patterns of Src and PI3-kinase (PI3K) activity. Toll receptors target Src activity to specific sites at the membrane, and Src recruits PI3K to the Toll-2 complex through tyrosine phosphorylation of the Toll-2 cytoplasmic domain. Reducing Src or PI3K activity disrupts planar-polarized myosin assembly, cell intercalation, and convergent extension, whereas constitutive Src activity promotes ectopic PI3K and myosin cortical localization. These results demonstrate that Toll receptors direct cell polarity and behavior by locally mobilizing Src and PI3K activity.