Intrinsic Relative Preference Profile of Pan-Kinase Inhibitor Drug Staurosporine towards the Clinically Occurring Gatekeeper Mutations in Protein Tyrosine Kinases

ABSTRACT Neisseria meningitidis, the causative agent of meningitis and septicemia, is able to attach to and invade a variety of cell types. In a previous study we showed that entry of N. meningitidis into human brain microvascular endothelial cells (HBMEC) is mediated by fibronectin bound to the outer membrane protein Opc, which forms a molecular bridge to α5β1-integrins. This interaction results in cytoskeletal remodeling and uptake of the bacteria. In this study we identified and characterized the intracellular signals involved in integrin-initiated uptake of N. meningitidis. We determined that the Src protein tyrosine kinases (PTKs) are activated in response to contact with N. meningitidis. Inhibition of Src PTK activity by the general tyrosine kinase inhibitor genistein and the specific Src inhibitor PP2 reduced Opc-mediated invasion of HBMEC and human embryonic kidney (HEK) 293T cells up to 90%. Moreover, overexpression of the cellular Src antagonist C-terminal Src kinase (CSK) also significantly reduced N. meningitidis invasion. Src PTK-deficient fibroblasts were impaired in the ability to internalize N. meningitidis and showed reduced phosphorylation of the cytoskeleton and decreased development of stress fibers. These data indicate that the Src family PTKs, particularly the Src protein, along with other proteins, are important signal proteins that are responsible for the transfer of signals from activated integrins to the cytoskeleton and thus mediate the endocytosis of N. meningitidis into brain endothelial cells.

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CGP 57148, a Tyrosine Kinase Inhibitor, Inhibits the Growth of Cells Expressing BCR-ABL, TEL-ABL, and TEL-PDGFR Fusion Proteins

Blood ◽

10.1182/blood.v90.12.4947 ◽

1997 ◽

Vol 90 (12) ◽

pp. 4947-4952 ◽

Cited By ~ 389

Author(s):

Martin Carroll ◽

Sayuri Ohno-Jones ◽

Shu Tamura ◽

Elisabeth Buchdunger ◽

Jürg Zimmermann ◽

...

Keyword(s):

Growth Factor ◽

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Kinase Activity ◽

Kinase Inhibitor ◽

Philadelphia Chromosome ◽

Lymphocytic Leukemia ◽

Protein Tyrosine Kinases ◽

Protein Tyrosine

Abstract CGP 57148 is a compound of the 2-phenylaminopyrimidine class that selectively inhibits the tyrosine kinase activity of the ABL and the platelet-derived growth factor receptor (PDGFR) protein tyrosine kinases. We previously showed that CGP 57148 selectively kills p210BCR-ABL–expressing cells. To extend these observations, we evaluated the ability of CGP 57148 to inhibit other activated ABL tyrosine kinases, including p185BCR-ABL and TEL-ABL. In cell-based assays of ABL tyrosine phosphorylation, inhibition of ABL kinase activity was observed at concentrations similar to that reported for p210BCR-ABL. Consistent with the in vitro profile of this compound, the growth of cells expressing activated ABL protein tyrosine kinases was inhibited in the absence of exogenous growth factor. Growth inhibition was also observed with a p185BCR-ABL–positive acute lymphocytic leukemia (ALL) cell line generated from a Philadelphia chromosome–positive ALL patient. As CGP 57148 inhibits the PDGFR kinase, we also showed that cells expressing an activated PDGFR tyrosine kinase, TEL-PDGFR, are sensitive to this compound. Thus, this compound may be useful for the treatment of a variety of BCR-ABL–positive leukemias and for treatment of the subset of chronic myelomonocytic leukemia patients with a TEL-PDGFR fusion protein.

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Activation of Protein Tyrosine Kinases byCoxiella burnetii: Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis

Infection and Immunity ◽

10.1128/iai.69.4.2520-2526.2001 ◽

2001 ◽

Vol 69 (4) ◽

pp. 2520-2526 ◽

Cited By ~ 29

Author(s):

Sonia Meconi ◽

Christian Capo ◽

Maryse Remacle-Bonnet ◽

Gilbert Pommier ◽

Didier Raoult ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Tyrosine Kinases ◽

Kinase Inhibitor ◽

Q Fever ◽

Protein Tyrosine Kinases ◽

Actin Reorganization ◽

Protein Tyrosine ◽

Cytoskeleton Organization ◽

Cytoskeleton Reorganization

ABSTRACT Coxiella burnetii, the agent of Q fever, is an obligate intracellular microorganism that grows in monocytes/macrophages. The internalization of virulent organisms by monocytes is lower than that of avirulent variants and is associated with actin cytoskeleton reorganization. We studied the activation of protein tyrosine kinases (PTKs) by C. burnetii in THP-1 monocytes. Virulent organisms induced early PTK activation and the tyrosine phosphorylation of several endogenous substrates, including Hck and Lyn, two Src-related kinases. PTK activation reflects C. burnetiivirulence since avirulent variants were unable to stimulate PTK. We also investigated the role of PTK activation in C. burnetii-stimulated F-actin reorganization. Tyrosine-phosphorylated proteins were colocalized with F-actin inside cell protrusions induced by C. burnetii, and PTK activity was increased in Triton X-100-insoluble fractions. In addition, lavendustin A, a PTK inhibitor, and PP1, a Src kinase inhibitor, prevented C. burnetii-induced cell protrusions and F-actin reorganization. We finally assessed the role of PTK activation in bacterial phagocytosis. Pretreatment of THP-1 cells with lavendustin A and PP1 upregulated the uptake of virulent C. burnetii but had no effect on the phagocytosis of avirulent organisms. Thus, it is likely that PTK activation by C. burnetii negatively regulates bacterial uptake by interfering with cytoskeleton organization.

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Haemophilus ducreyi LspA Proteins Are Tyrosine Phosphorylated by Macrophage-Encoded Protein Tyrosine Kinases

Infection and Immunity ◽

10.1128/iai.00513-08 ◽

2008 ◽

Vol 76 (10) ◽

pp. 4692-4702 ◽

Cited By ~ 8

Author(s):

Kaiping Deng ◽

Jason R. Mock ◽

Steven Greenberg ◽

Nicolai S. C. van Oers ◽

Eric J. Hansen

Keyword(s):

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Immune Cell ◽

Kinase Inhibitor ◽

Divalent Cations ◽

Site Directed Mutagenesis ◽

Haemophilus Ducreyi ◽

Protein Tyrosine Kinases ◽

Protein Tyrosine

ABSTRACTThe LspA proteins (LspA1 and LspA2) ofHaemophilus ducreyiare necessary for this pathogen to inhibit the phagocytic activity of macrophage cell lines, an event that can be correlated with a reduction in the level of active Src family protein tyrosine kinases (PTKs) in these eukaryotic cells. During studies investigating this inhibitory mechanism, it was discovered that the LspA proteins themselves were tyrosine phosphorylated after wild-typeH. ducreyicells were incubated with macrophages. LspA proteins in cell-free concentratedH. ducreyiculture supernatant fluid could also be tyrosine phosphorylated by macrophages. This ability to tyrosine phosphorylate the LspA proteins was not limited to immune cell lineages but could be accomplished by both HeLa and COS-7 cells. Kinase inhibitor studies with macrophages demonstrated that the Src family PTKs were required for this tyrosine phosphorylation activity. In silico methods and site-directed mutagenesis were used to identify EPIYG and EPVYA motifs in LspA1 that contained tyrosines that were targets for phosphorylation. A total of four tyrosines could be phosphorylated in LspA1, with LspA2 containing eight predicted tyrosine phosphorylation motifs. Purified LspA1 fusion proteins containing either the EPIYG or EPVYA motifs were shown to be phosphorylated by purified Src PTK in vitro. Macrophage lysates could also tyrosine phosphorylate the LspA proteins and an LspA1 fusion protein via a mechanism that was dependent on the presence of both divalent cations and ATP. Several motifs known to interact with or otherwise affect eukaryotic kinases were identified in the LspA proteins.

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CGP 57148, a Tyrosine Kinase Inhibitor, Inhibits the Growth of Cells Expressing BCR-ABL, TEL-ABL, and TEL-PDGFR Fusion Proteins

Blood ◽

10.1182/blood.v90.12.4947.4947_4947_4952 ◽

1997 ◽

Vol 90 (12) ◽

pp. 4947-4952 ◽

Cited By ~ 18

Author(s):

Martin Carroll ◽

Sayuri Ohno-Jones ◽

Shu Tamura ◽

Elisabeth Buchdunger ◽

Jürg Zimmermann ◽

...

Keyword(s):

Growth Factor ◽

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Kinase Activity ◽

Kinase Inhibitor ◽

Philadelphia Chromosome ◽

Lymphocytic Leukemia ◽

Protein Tyrosine Kinases ◽

Protein Tyrosine

CGP 57148 is a compound of the 2-phenylaminopyrimidine class that selectively inhibits the tyrosine kinase activity of the ABL and the platelet-derived growth factor receptor (PDGFR) protein tyrosine kinases. We previously showed that CGP 57148 selectively kills p210BCR-ABL–expressing cells. To extend these observations, we evaluated the ability of CGP 57148 to inhibit other activated ABL tyrosine kinases, including p185BCR-ABL and TEL-ABL. In cell-based assays of ABL tyrosine phosphorylation, inhibition of ABL kinase activity was observed at concentrations similar to that reported for p210BCR-ABL. Consistent with the in vitro profile of this compound, the growth of cells expressing activated ABL protein tyrosine kinases was inhibited in the absence of exogenous growth factor. Growth inhibition was also observed with a p185BCR-ABL–positive acute lymphocytic leukemia (ALL) cell line generated from a Philadelphia chromosome–positive ALL patient. As CGP 57148 inhibits the PDGFR kinase, we also showed that cells expressing an activated PDGFR tyrosine kinase, TEL-PDGFR, are sensitive to this compound. Thus, this compound may be useful for the treatment of a variety of BCR-ABL–positive leukemias and for treatment of the subset of chronic myelomonocytic leukemia patients with a TEL-PDGFR fusion protein.

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Role of Protein Tyrosine Kinase p53/56lyn in Diminished Lipopolysaccharide Priming of Formylmethionylleucyl- phenylalanine-Induced Superoxide Production in Human Newborn Neutrophils

Infection and Immunity ◽

10.1128/iai.72.11.6455-6462.2004 ◽

2004 ◽

Vol 72 (11) ◽

pp. 6455-6462 ◽

Cited By ~ 13

Author(s):

Sen Rong Yan ◽

David M. Byers ◽

Robert Bortolussi

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Protein Tyrosine Kinase ◽

Kinase Inhibitor ◽

Critical Role ◽

Polymorphonuclear Neutrophils ◽

Protein Tyrosine Kinases ◽

Protein Tyrosine ◽

Adult Cells

ABSTRACT Human newborns are more susceptible than adults to bacterial infection. With gram-negative bacteria, this may be due to a diminished response of newborn leukocytes to lipopolysaccharide (LPS). Since protein tyrosine kinase inhibition abolishes LPS priming in adult cells, we hypothesized that protein tyrosine kinases may have a critical role in LPS priming of polymorphonuclear neutrophils (PMNs) and that newborn PMNs may have altered protein tyrosine kinase activities. In the present study, we investigated the role of src family protein tyrosine kinases in the LPS response of newborn PMNs compared to adult cells. In a respiratory assay, the LPS-primed increase in formylmethionylleucylphenylalanine (fMLP)-triggered O2 − release by adult PMNs was greatly decreased by PP1 [4-amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine], a src kinase inhibitor, to the level of untreated newborn PMNs, in which LPS failed to prime. LPS activated the src-like kinases p59hck (HCK) and p58fgr (FGR) in both adult and newborn PMNs but increased the activation of p53/56 lyn (LYN) only in adult cells. In newborn PMNs, LYN was highly phosphorylated independent of LPS. We evaluated subcellular fractions of PMNs and found that the phosphorylated form of LYN was mainly in the Triton-extractable, cytosolic fraction in adult PMNs, while in newborn cells it was located mainly in Triton-insoluble, granule- and membrane-associated fractions. In contrast, the phosphorylated mitogen-activated protein kinases ERK1/2 and p38 were mainly detected in the cytosol in both adult and newborn PMNs. These data indicate a role for LYN in the regulation of LPS priming. The trapping of phosphorylated LYN in the membrane-granule fraction in newborn PMNs may contribute to the deficiency of newborn cells in responding to LPS stimulation.

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