Measuring Phosphorylation-Specific Changes in Response to Kinase Inhibitors in Mammalian Cells Using Quantitative Proteomics

2011 ◽  
pp. 217-231 ◽  
Author(s):  
Nurhan Özlü ◽  
Marc Kirchner ◽  
Judith Jebanathirajah Steen
Keyword(s):  
Quantitative Proteomics ◽  
Mammalian Cells ◽  
Kinase Inhibitors

scholarly journals Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature

2017 ◽  
Vol 216 (11) ◽  
pp. 3745-3765 ◽  
Author(s):  
Frederic Daste ◽  
Astrid Walrant ◽  
Mikkel R. Holst ◽  
Jonathan R. Gadsby ◽  
Julia Mason ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Actin Polymerization ◽  
Kinase Inhibitors ◽  
Mammalian Cell Culture ◽  
Membrane Curvature ◽  
Coated Pits ◽  
Bar Domain ◽  
Oculocerebrorenal Syndrome ◽  
Cascade Amplification ◽  
Hydrolysis Of

The conditional use of actin during clathrin-mediated endocytosis in mammalian cells suggests that the cell controls whether and how actin is used. Using a combination of biochemical reconstitution and mammalian cell culture, we elucidate a mechanism by which the coincidence of PI(4,5)P2 and PI(3)P in a curved vesicle triggers actin polymerization. At clathrin-coated pits, PI(3)P is produced by the INPP4A hydrolysis of PI(3,4)P2, and this is necessary for actin-driven endocytosis. Both Cdc42⋅guanosine triphosphate and SNX9 activate N-WASP–WIP- and Arp2/3-mediated actin nucleation. Membrane curvature, PI(4,5)P2, and PI(3)P signals are needed for SNX9 assembly via its PX–BAR domain, whereas signaling through Cdc42 is activated by PI(4,5)P2 alone. INPP4A activity is stimulated by high membrane curvature and synergizes with SNX9 BAR domain binding in a process we call curvature cascade amplification. We show that the SNX9-driven actin comets that arise on human disease–associated oculocerebrorenal syndrome of Lowe (OCRL) deficiencies are reduced by inhibiting PI(3)P production, suggesting PI(3)P kinase inhibitors as a therapeutic strategy in Lowe syndrome.

scholarly journals Mechanotransduction in an extracted cell model: Fyn drives stretch- and flow-elicited PECAM-1 phosphorylation

2008 ◽  
Vol 182 (4) ◽  
pp. 753-763 ◽  
Author(s):  
Yi-Jen Chiu ◽  
Elena McBeath ◽  
Keigi Fujiwara
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Adhesion Molecule ◽  
Mammalian Cells ◽  
Kinase Inhibitors ◽  
Vascular Endothelial Cells ◽  
Cell Model ◽  
Vascular Endothelial ◽  
Small Interfering Rnas ◽  
Essential Components

Mechanosensing followed by mechanoresponses by cells is well established, but the mechanisms by which mechanical force is converted into biochemical events are poorly understood. Vascular endothelial cells (ECs) exhibit flow- and stretch-dependent responses and are widely used as a model for studying mechanotransduction in mammalian cells. Platelet EC adhesion molecule 1 (PECAM-1) is tyrosine phosphorylated when ECs are exposed to flow or when PECAM-1 is directly pulled, suggesting that it is a mechanochemical converter. We show that PECAM-1 phosphorylation occurs when detergent-extracted EC monolayers are stretched, indicating that this phosphorylation is mechanically triggered and does not require the intact plasma membrane and soluble cytoplasmic components. Using kinase inhibitors and small interfering RNAs, we identify Fyn as the PECAM-1 kinase associated with the model. We further show that stretch- and flow-induced PECAM-1 phosphorylation in intact ECs is abolished when Fyn expression is down-regulated. We suggest that PECAM-1 and Fyn are essential components of a PECAM-1–based mechanosensory complex in ECs.

scholarly journals Comparative assessment of the effects of bumped kinase inhibitors on early zebrafish embryo development and pregnancy in mice

2020 ◽  
Author(s):  
Nicoleta Anghel ◽  
Pablo A. Winzer ◽  
Dennis Imhof ◽  
Joachim Müller ◽  
Javier Langa ◽  
...  
Keyword(s):  
Pregnancy Outcome ◽  
Embryo Development ◽  
Mammalian Cells ◽  
Zebrafish Embryo ◽  
Kinase Inhibitors ◽  
Corn Oil ◽  
Neospora Caninum ◽  
Clear Correlation ◽  
Related Factors ◽  
The Impact

AbstractBumped kinase inhibitors (BKIs) are effective against a variety of apicomplexan parasites. Fifteen BKIs with promising in vitro efficacy against Neospora caninum tachyzoites, low cytotoxicity in mammalian cells, and no toxic effects in non-pregnant BALB/c mice, were assessed in pregnant mice. Drugs were emulsified in corn oil and applied by gavage for 5 days. Five BKIs did not affect pregnancy, 5 BKIs exhibited 15-35% of neonatal mortality, and 5 compounds caused strong effects (infertility, abortion, stillbirth and pup mortality). Additionally, the impact of these compounds on zebrafish (Danio rerio) embryo development was assessed by exposing freshly fertilized eggs to 0.2-50μM of BKIs and microscopical monitoring of embryo development in a blinded manner during 4 days. We propose an algorithm that includes quantification of malformations and embryo deaths, and established a scoring system that allows to calculate an impact score (Si) that indicates at which concentrations BKIs visibly affect zebrafish embryo development. Comparison of the two models showed that for 9 compounds no clear correlation between Si and pregnancy outcome was visible. However, those 3 BKIs affecting zebrafish embryos only at high concentrations (40μM or higher) did not impair mouse pregnancy at all, and those 3 compounds that inhibited zebrafish embryo development already at 0.2μM showed detrimental effects in the pregnancy model. Thus, the zebrafish embryo development test has a limited predictive value to foresee pregnancy outcome in BKI-treated mice. We conclude, that maternal health-related factors such as cardiovascular, pharmacokinetic and/or bioavailability properties also contribute to BKI-pregnancy effects.

scholarly journals Arf-1 (ADP-ribosylation factor-1) is involved in the activation of a mammalian Na+-selective current

2004 ◽  
Vol 377 (2) ◽  
pp. 539-544 ◽  
Author(s):  
Sebastian STRAUBE ◽  
Anant B. PAREKH
Keyword(s):  
Mammalian Cells ◽  
Kinase Inhibitors ◽  
Brefeldin A ◽  
Cell Types ◽  
Protein Kinase Inhibitors ◽  
Na Current ◽  
Camp Phosphodiesterase ◽  
Pipette Solution ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

Stimulation of mammalian cells often results in an increase in the intracellular Na+ concentration, brought about by Na+ influx into the cell via Na+-permeable ion channels. In some cell types, particularly renal epithelia and mast cells, non-hydrolysable analogues of GTP, such as GTP[S] (guanosine 5´-[γ-thio]triphosphate), activate a non-voltage-activated Na+-selective current. We have carried out whole-cell patch–clamp experiments to examine how GTP[S] activates the Na+ current in a rat mast cell line. The ability of GTP[S] to activate Na+ influx was prevented by including GTP in the pipette solution, indicating the involvement of small G-proteins. Brefeldin A and Arf-1-(2–17), inhibitors of Arf-1 (ADP-ribosylation factor-1) proteins, suppressed the activation of Na+ entry by GTP[S]. However, non-active succinylated Arf-1-(2–17) or an N-terminal myristoylated peptide directed towards Arf-5 were ineffective. Arf proteins modulate the cytoskeleton, and disruption of the cytoskeleton with cytochalasin D or its stabilization with phalloidin impaired the development of the Na+ current. Disaggregation of microtubules was without effect. Dialysis with cAMP or inhibition of cAMP phosphodiesterase with caffeine both decreased the extent of Na+ entry, and this was not prevented by pre-treatment with broad-spectrum protein kinase inhibitors. Collectively, our results suggest that the mechanism of activation of a mammalian non-voltage-activated Na+-selective current requires an Arf small G-protein, most probably Arf-1.

scholarly journals RRP1B Targets PP1 to Mammalian Cell Nucleoli and Is Associated with Pre-60S Ribosomal Subunits

2010 ◽  
Vol 21 (23) ◽  
pp. 4212-4226 ◽  
Author(s):  
Delphine Chamousset ◽  
Veerle De Wever ◽  
Greg B. Moorhead ◽  
Yan Chen ◽  
Francois-Michel Boisvert ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Quantitative Proteomics ◽  
Mammalian Cells ◽  
Ribosome Biogenesis ◽  
Large Fraction ◽  
Ribosomal Subunit ◽  
Growth Conditions ◽  
Live Cells ◽  
Ribosomal Subunits ◽  
Subnuclear Localization

A pool of protein phosphatase 1 (PP1) accumulates within nucleoli and accounts for a large fraction of the serine/threonine protein phosphatase activity in this subnuclear structure. Using a combination of fluorescence imaging with quantitative proteomics, we mapped the subnuclear localization of the three mammalian PP1 isoforms stably expressed as GFP-fusions in live cells and identified RRP1B as a novel nucleolar targeting subunit that shows a specificity for PP1β and PP1γ. RRP1B, one of two mammalian orthologues of the yeast Rrp1p protein, shows an RNAse-dependent localization to the granular component of the nucleolus and distributes in a similar manner throughout the cell cycle to proteins involved in later steps of rRNA processing. Quantitative proteomic analysis of complexes containing both RRP1B and PP1γ revealed enrichment of an overlapping subset of large (60S) ribosomal subunit proteins and pre-60S nonribosomal proteins involved in mid-late processing. Targeting of PP1 to this complex by RRP1B in mammalian cells is likely to contribute to modulation of ribosome biogenesis by mechanisms involving reversible phosphorylation events, thus playing a role in the rapid transduction of cellular signals that call for regulation of ribosome production in response to cellular stress and/or changes in growth conditions.

scholarly journals Sphingomyelin Metabolism Is a Regulator of K-Ras Function

2017 ◽  
Vol 38 (3) ◽  
Author(s):  
Dharini van der Hoeven ◽  
Kwang-jin Cho ◽  
Yong Zhou ◽  
Xiaoping Ma ◽  
Wei Chen ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Mammalian Cells ◽  
Tricyclic Antidepressants ◽  
Kinase Inhibitors ◽  
Acid Sphingomyelinase ◽  
Pharmacological Agents ◽  
Content Type ◽  
C Elegans ◽  
Pancreatic Cancer Cells ◽  
Genes Encoding

ABSTRACTK-Ras must localize to the plasma membrane (PM) for biological activity. We show here that multiple acid sphingomyelinase (ASM) inhibitors, including tricyclic antidepressants, mislocalized phosphatidylserine (PtdSer) and K-RasG12V from the PM, resulting in abrogation of K-RasG12V signaling and potent, selective growth inhibition of mutant K-Ras-transformed cancer cells. Concordantly, in nude mice, the ASM inhibitor fendiline decreased the rate of growth of oncogenic K-Ras-expressing MiaPaCa-2 tumors but had no effect on the growth of the wild-type K-Ras-expressing BxPC-3 tumors. ASM inhibitors also inhibited activated LET-60 (a K-Ras ortholog) signaling inCaenorhabditis elegans, as evidenced by suppression of the induced multivulva phenotype. Using RNA interference againstC. elegansgenes encoding other enzymes in the sphingomyelin (SM) biosynthetic pathway, we identified 14 enzymes whose knockdown strongly or moderately suppressed the LET-60 multivulva phenotype. In mammalian cells, pharmacological agents that target these enzymes all depleted PtdSer from the PM and caused K-RasG12V mislocalization. These effects correlated with changes in SM levels or subcellular distribution. Selected compounds, including sphingosine kinase inhibitors, potently inhibited the proliferation of oncogenic K-Ras-expressing pancreatic cancer cells. In conclusion, these results show that normal SM metabolism is critical for K-Ras function, which may present therapeutic options for the treatment of K-Ras-driven cancers.

scholarly journals A High-Content Screening Assay for Small-Molecule Modulators of Oncogene-Induced Senescence

2013 ◽  
Vol 18 (9) ◽  
pp. 1054-1061 ◽  
Author(s):  
Benjamin G. Bitler ◽  
Lauren S. Fink ◽  
Zhi Wei ◽  
Jeffrey R. Peterson ◽  
Rugang Zhang
Keyword(s):  
Small Molecule ◽  
Cellular Senescence ◽  
Mammalian Cells ◽  
Kinase Inhibitors ◽  
Imaging System ◽  
Human Fibroblasts ◽  
High Content Screening ◽  
Screening Assay ◽  
Critical Pathway ◽  
Inhibition Of Proliferation

Cellular senescence is a state of stable cell growth arrest. Activation of oncogenes such as RAS in mammalian cells typically triggers cellular senescence. Oncogene-induced senescence (OIS) is an important tumor suppression mechanism, and suppression of OIS contributes to cell transformation. Oncogenes trigger senescence through a multitude of incompletely understood downstream signaling events that frequently involve protein kinases. To identify target proteins required for RAS-induced senescence, we developed a small-molecule screen in primary human fibroblasts undergoing senescence induced by oncogenic RAS (H-RasG12V). Using a high-content imaging system to monitor two hallmarks of senescence, senescence-associated β-galactosidase activity expression and inhibition of proliferation, we screened a library of known small-molecule kinase inhibitors for those that suppressed OIS. Identified compounds were subsequently validated and confirmed using a third marker of senescence, senescence-associated heterochromatin foci. In summary, we have established a novel high-content screening platform that may be useful for elucidating signaling pathways mediating OIS by targeting critical pathway components.

scholarly journals Development of novel anti-malarial from structurally diverse library of molecules, targeting plant-like CDPK1, a multistage growth regulator of P. falciparum

2020 ◽  
Vol 477 (10) ◽  
pp. 1951-1970 ◽  
Author(s):  
Ravi Jain ◽  
Sakshi Gupta ◽  
Manoj Munde ◽  
Soumya Pati ◽  
Shailja Singh
Keyword(s):  
Mammalian Cells ◽  
Kinase Inhibitors ◽  
Binding Constants ◽  
Binding Pocket ◽  
Atp Binding ◽  
Parasite Life Cycle ◽  
Dependent Protein ◽  
Non Covalent Interactions ◽  
Low K

Upon Plasmodium falciparum merozoites exposure to low [K+] environment in blood plasma, there is escalation of cytosolic [Ca2+] which activates Ca2+-Dependent Protein Kinase 1 (CDPK1), a signaling hub of intra-erythrocytic proliferative stages of parasite. Given its high abundance and multidimensional attributes in parasite life-cycle, this is a lucrative target for designing antimalarials. Towards this, we have virtually screened MyriaScreenII diversity collection of 10,000 drug-like molecules, which resulted in 18 compounds complementing ATP-binding pocket of CDPK1. In vitro screening for toxicity in mammalian cells revealed that these compounds are non-toxic in nature. Furthermore, SPR analysis demonstrated differential binding affinity of these compounds towards recombinantly purified CDPK1 protein. Selection of lead compound 1 was performed by evaluating their inhibitory effects on phosphorylation and ATP binding activities of CDPK1. Furthermore, in vitro biophysical evaluations by ITC and FS revealed that binding of compound 1 is driven by formation of energetically favorable non-covalent interactions, with different binding constants in presence and absence of Ca2+, and TSA authenticated stability of compound 1 bound CDPK1 complex. Finally, compound 1 strongly inhibited intra-erythrocytic growth of P. falciparum in vitro. Conceivably, we propose a novel CDPK1-selective inhibitor, step towards developing pan-CDPK kinase inhibitors, prerequisite for cross-stage anti-malarial protection.

scholarly journals New Class of Competitive Inhibitor of Bacterial Histidine Kinases

2005 ◽  
Vol 187 (23) ◽  
pp. 8196-8200 ◽  
Author(s):  
Raymond Gilmour ◽  
J. Estelle Foster ◽  
Qin Sheng ◽  
Jonathan R. McClain ◽  
Anna Riley ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Histidine Kinase ◽  
Mammalian Cells ◽  
Kinase Inhibitors ◽  
Competitive Inhibitor ◽  
Histidine Kinases ◽  
New Class ◽  
Starting Compound ◽  
New Antibiotics ◽  
Specific Inhibitors

ABSTRACT Bacterial histidine kinases have been proposed as targets for the discovery of new antibiotics, yet few specific inhibitors of bacterial histidine kinases have been reported. We report here a novel thienopyridine (TEP) compound that inhibits bacterial histidine kinases competitively with respect to ATP but does not comparably inhibit mammalian serine/threonine kinases. Although it partitions into membranes and does not inhibit the growth of bacterial or mammalian cells, TEP could serve as a starting compound for a new class of histidine kinase inhibitors with antibacterial activity.

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