A mutation at the ATP-binding site of pp60v-src abolishes kinase activity, transformation, and tumorigenicity

1985 ◽  
Vol 5 (7) ◽  
pp. 1772-1779
Author(s):  
M A Snyder ◽  
J M Bishop ◽  
J P McGrath ◽  
A D Levinson
Keyword(s):  
Binding Site ◽  
Kinase Activity ◽  
Atp Binding ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Atp Binding Site ◽  
Specific Immunoglobulin ◽  
Dependent Protein ◽  
Infected Cells

We constructed a mutant, called RSV-SF2, at the ATP-binding site of pp60v-src. In this mutant, lysine-295 is replaced with methionine. SF2 pp60v-src was found to have a half-life similar to that of wild-type pp60v-src and was localized in the membranous fraction of the cell. Rat cells expressing SF2 pp60v-src were morphologically untransformed and do not form tumors. The SF2 pp60v-src isolated from these cells lacked kinase activity with either specific immunoglobulin or other substrates, and expression of SF2 pp60v-src failed to cause an increase of total phosphotyrosine in the proteins of infected cells. Wild-type pp60v-src was phosphorylated on serine and tyrosine in infected cells, and the analogous phosphorylations could also be carried out in vitro. Phosphorylation of serine was catalyzed by a cyclic AMP-dependent protein kinase, and phosphorylation of tyrosine was perhaps catalyzed by pp60v-src itself. By contrast, SF2 pp60v-src could not be phosphorylated on serine or tyrosine either in infected cells or in vitro. These findings strengthen the belief that the phosphotransferase activity of pp60v-src is required for neoplastic transformation by the protein and suggest that the binding of ATP to pp60v-src elicits an allosteric change required for phosphorylation of serine in the protein.

scholarly journals A mutation at the ATP-binding site of pp60v-src abolishes kinase activity, transformation, and tumorigenicity.

1985 ◽  
Vol 5 (7) ◽  
pp. 1772-1779 ◽  
Author(s):  
M A Snyder ◽  
J M Bishop ◽  
J P McGrath ◽  
A D Levinson
Keyword(s):  
Binding Site ◽  
Kinase Activity ◽  
Atp Binding ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Atp Binding Site ◽  
Specific Immunoglobulin ◽  
Dependent Protein ◽  
Infected Cells

We constructed a mutant, called RSV-SF2, at the ATP-binding site of pp60v-src. In this mutant, lysine-295 is replaced with methionine. SF2 pp60v-src was found to have a half-life similar to that of wild-type pp60v-src and was localized in the membranous fraction of the cell. Rat cells expressing SF2 pp60v-src were morphologically untransformed and do not form tumors. The SF2 pp60v-src isolated from these cells lacked kinase activity with either specific immunoglobulin or other substrates, and expression of SF2 pp60v-src failed to cause an increase of total phosphotyrosine in the proteins of infected cells. Wild-type pp60v-src was phosphorylated on serine and tyrosine in infected cells, and the analogous phosphorylations could also be carried out in vitro. Phosphorylation of serine was catalyzed by a cyclic AMP-dependent protein kinase, and phosphorylation of tyrosine was perhaps catalyzed by pp60v-src itself. By contrast, SF2 pp60v-src could not be phosphorylated on serine or tyrosine either in infected cells or in vitro. These findings strengthen the belief that the phosphotransferase activity of pp60v-src is required for neoplastic transformation by the protein and suggest that the binding of ATP to pp60v-src elicits an allosteric change required for phosphorylation of serine in the protein.

scholarly journals Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase

1984 ◽  
Vol 220 (3) ◽  
pp. 677-683 ◽  
Author(s):  
J E Kudlow ◽  
Y Leung
Keyword(s):  
Protein Kinase ◽  
Binding Site ◽  
Egf Receptor ◽  
Light Exposure ◽  
Atp Binding ◽  
Receptor Kinase ◽  
Dependent Protein Kinase ◽  
Atp Binding Site ◽  
Dependent Protein ◽  
Epidermal Growth

Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analogue of ATP, 3′-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5′-triphosphate (arylazido-beta-alanyl-ATP) on the EGF-dependent protein kinase in A431 human tumour cell plasma membrane vesicles was investigated. This analogue was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analogue concentration of 1 microM and complete inactivation occurred at 10 microM when a 2 min light exposure was used. Arylazido-beta-alanine at 100 microM and ATP at 100 microM were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analogue could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5′-[beta gamma-imido]triphosphate or 20 mM-guanosine 5′-[beta gamma-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-beta-alanyl-[alpha-32P]ATP was capable of labelling membrane proteins in a photodependent manner. Numerous proteins were labelled, the most prominent of which ran with an apparent Mr of 53000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of Mr corresponding to the EGF receptor (170000). Immunoprecipitation of affinity-labelled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the Mr 170000 receptor protein was photoaffinity labelled by the analogue. The Mr 53000 peptide was not specifically bound by the anti-receptor antibody. The affinity labelling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. These studies demonstrate that arylazido-beta-alanyl-ATP interacts with the ATP-binding site of the EGF-receptor kinase with apparent high affinity and that this analogue is an effective photoaffinity label for the kinase. Furthermore, these studies demonstrate that the EGF receptor, identified by using monoclonal antibodies, contains an ATP-binding site, providing further confirmation that the EGF receptor and EGF-dependent protein kinase are domains of the Mr 170000 protein.

scholarly journals Contribution of the ATP Binding Site of ParE to Susceptibility to Novobiocin and Quinolones in Streptococcus pneumoniae

2005 ◽  
Vol 187 (4) ◽  
pp. 1536-1540 ◽  
Author(s):  
Philippe Dupont ◽  
Alexandra Aubry ◽  
Emmanuelle Cambau ◽  
Laurent Gutmann
Keyword(s):  
Streptococcus Pneumoniae ◽  
Binding Site ◽  
Quinolone Resistance ◽  
Atp Binding ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Atp Binding Site ◽  
Open Conformation ◽  
A Subunit

ABSTRACT In Streptococcus pneumoniae, an H103Y substitution in the ATP binding site of the ParE subunit of topoisomerase IV was shown to confer quinolone resistance and hypersensitivity to novobiocin when associated with an S84F change in the A subunit of DNA gyrase. We reconstituted in vitro the wild-type topoisomerase IV and its ParE mutant. The ParE mutant enzyme showed a decreased activity for decatenation at subsaturating ATP levels and was more sensitive to inhibition by novobiocin but was as sensitive to quinolones. These results show that the ParE alteration H103Y alone is not responsible for quinolone resistance and agree with the assumption that it facilitates the open conformation of the ATP binding site that would lead to novobiocin hypersensitivity and to a higher requirement of ATP.

scholarly journals Role of the Glycine Triad in the ATP-binding Site of cAMP-dependent Protein Kinase

1997 ◽  
Vol 272 (27) ◽  
pp. 16946-16954 ◽  
Author(s):  
Wolfram Hemmer ◽  
Maria McGlone ◽  
Igor Tsigelny ◽  
Susan S. Taylor
Keyword(s):  
Protein Kinase ◽  
Binding Site ◽  
Atp Binding ◽  
Dependent Protein Kinase ◽  
Atp Binding Site ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

The JAK2 Kinase Inhibitor LS104 Induces Growth-Arrest and Apoptosis in JAK2V617F Positive Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3544-3544 ◽  
Author(s):  
Daniel B. Lipka ◽  
Linda S. Hoffmann ◽  
Florian Heidel ◽  
Boyka Markova ◽  
Stefan Kasper ◽  
...  
Keyword(s):  
Binding Site ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Erythropoietin Receptor ◽  
Kinase Assay ◽  
Atp Binding ◽  
Jak Inhibitor ◽  
Atp Binding Site ◽  
V617f Mutation

Abstract The JAK2V617F-mutation (V617F) is a novel, highly prevalent molecular marker in Ph-negative myeloproliferative disease (MPD). In vitro, the V617F mutation confers cytokine independent growth of Ba/F3 cells expressing erythropoietin receptor (EpoR) and constitutive activation of the JAK2 kinase and of the JAK-STAT pathway. In a murine bone-marrow transplant model the V617F-mutation alone is sufficient to induce a polycythemia vera-like phenotype. Therefore, mutant JAK2 kinase is a promising target for kinase inhibitor development. In this report, we characterize the small molecule LS104 (previously CR4; Grunberger et al., Blood 2003) as a novel non-ATP-competitive JAK2V617F kinase inhibitor. Ba/F3 cells stably transfected with EpoR and either the V617F-mutant (Ba/F3-EpoR-VF) or wildtype JAK2 (Ba/F3-EpoR-WT) were treated with LS104. Apoptosis assays as well as immunoblotting of JAK2 and of downstream signalling pathways were performed. The effects of LS104 on kinase activity were determined in an in vitro JAK2 kinase assay. A combination of LS104 with JAK-inhibitor I, which acts via the ATP-binding site, was tested in apoptosis assays. Finally, growth of endogenous erythroid colonies (EECs) obtained from patients with V617F-positive MPD was tested upon addition of LS104. LS104 selectively and dose dependently induced apoptosis in Ba/F3-EpoR-VF cells as compared to Ba/F3-EpoR-WT control cells. By immunoblotting we found inhibition of JAK2 autophosphorylation and of downstream targets as STAT5, AKT and ERK upon treatment with LS104. Activation of these targets by JAK2 was confirmed in experiments using JAK2 siRNA. The IC50 for JAK2 in an in vitro kinase assay using LS104 was <5μM, and this effect was not reversible using increased ATP-doses. Combination treatment of Ba/F3-EpoR-VF cells using LS104 plus JAK-inhibitor I lead to significantly increased apoptosis as compared to each substance alone. Furthermore, we observed 89% inhibition of in vitro formation of EECs at 10μM LS104 whereas growth of CFU-GM obtained from normal controls was virtually unaffected. Taken together, our data show that LS104 specifically inhibits JAK2 kinase activity and JAK2 downstream signals and thereby induces apoptosis in V617F-positive cells. Our data suggest, that LS104 either irreversibly binds to the ATP-binding site or acts as a substrate kinase inhibitor and may be combined with ATP-competitive JAK2 inhibitors to enhance treatment efficacy. Growth of EECs from patients with MPD is shown to be significantly suppressed by LS104. Based on these data, a phase I/II clinical trial of LS104 for patients with JAK2V617F-positive MPD has been initiated recently.

New Mechanistic Insight on the PIM-1 Kinase Inhibitor AZD1208 Using Multidrug Resistant Human Erythroleukemia Cell Lines and Molecular Docking Simulations

2019 ◽  
Vol 19 (11) ◽  
pp. 914-926 ◽  
Author(s):  
Maiara Bernardes Marques ◽  
Michael González-Durruthy ◽  
Bruna Félix da Silva Nornberg ◽  
Bruno Rodrigues Oliveira ◽  
Daniela Volcan Almeida ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Binding Site ◽  
Cell Lines ◽  
Chemotherapeutic Agents ◽  
Atp Binding ◽  
Human Leukemia ◽  
Atp Binding Site ◽  
Mechanistic Insight ◽  
Multiple Drugs

Background:PIM-1 is a kinase which has been related to the oncogenic processes like cell survival, proliferation, and multidrug resistance (MDR). This kinase is known for its ability to phosphorylate the main extrusion pump (ABCB1) related to the MDR phenotype.Objective:In the present work, we tested a new mechanistic insight on the AZD1208 (PIM-1 specific inhibitor) under interaction with chemotherapy agents such as Daunorubicin (DNR) and Vincristine (VCR).Materials and Methods:In order to verify a potential cytotoxic effect based on pharmacological synergism, two MDR cell lines were used: Lucena (resistant to VCR) and FEPS (resistant to DNR), both derived from the K562 non-MDR cell line, by MTT analyses. The activity of Pgp was ascertained by measuring accumulation and the directional flux of Rh123. Furthermore, we performed a molecular docking simulation to delve into the molecular mechanism of PIM-1 alone, and combined with chemotherapeutic agents (VCR and DNR).Results:Our in vitro results have shown that AZD1208 alone decreases cell viability of MDR cells. However, co-exposure of AZD1208 and DNR or VCR reverses this effect. When we analyzed the ABCB1 activity AZD1208 alone was not able to affect the pump extrusion. Differently, co-exposure of AZD1208 and DNR or VCR impaired ABCB1 activity, which could be explained by compensatory expression of abcb1 or other extrusion pumps not analyzed here. Docking analysis showed that AZD1208 is capable of performing hydrophobic interactions with PIM-1 ATP- binding-site residues with stronger interaction-based negative free energy (FEB, kcal/mol) than the ATP itself, mimicking an ATP-competitive inhibitory pattern of interaction. On the same way, VCR and DNR may theoretically interact at the same biophysical environment of AZD1208 and also compete with ATP by the PIM-1 active site. These evidences suggest that AZD1208 may induce pharmacodynamic interaction with VCR and DNR, weakening its cytotoxic potential in the ATP-binding site from PIM-1 observed in the in vitro experiments.Conclusion:Finally, the current results could have a pre-clinical relevance potential in the rational polypharmacology strategies to prevent multiple-drugs resistance in human leukemia cancer therapy.

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