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1.
Oncogene ; 33(12): 1590-600, 2014 Mar 20.
Article in English | MEDLINE | ID: mdl-23542178

ABSTRACT

The mammalian target of rapamycin (mTOR) regulates cell growth by integrating nutrient and growth factor signaling and is strongly implicated in cancer. But mTOR is not an oncogene, and which tumors will be resistant or sensitive to new adenosine triphosphate (ATP) competitive mTOR inhibitors now in clinical trials remains unknown. We screened a panel of over 600 human cancer cell lines to identify markers of resistance and sensitivity to the mTOR inhibitor PP242. RAS and phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutations were the most significant genetic markers for resistance and sensitivity to PP242, respectively; colon origin was the most significant marker for resistance based on tissue type. Among colon cancer cell lines, those with KRAS mutations were most resistant to PP242, whereas those without KRAS mutations most sensitive. Surprisingly, cell lines with co-mutation of PIK3CA and KRAS had intermediate sensitivity. Immunoblot analysis of the signaling targets downstream of mTOR revealed that the degree of cellular growth inhibition induced by PP242 was correlated with inhibition of phosphorylation of the translational repressor eIF4E-binding protein 1 (4E-BP1), but not ribosomal protein S6 (rpS6). In a tumor growth inhibition trial of PP242 in patient-derived colon cancer xenografts, resistance to PP242-induced inhibition of 4E-BP1 phosphorylation and xenograft growth was again observed in KRAS mutant tumors without PIK3CA co-mutation, compared with KRAS wild-type controls. We show that, in the absence of PIK3CA co-mutation, KRAS mutations are associated with resistance to PP242 and that this is specifically linked to changes in the level of phosphorylation of 4E-BP1.


Subject(s)
Adenosine Triphosphate/metabolism , Antineoplastic Agents/pharmacology , Binding, Competitive/drug effects , Carrier Proteins/metabolism , Drug Resistance, Neoplasm/drug effects , Indoles/pharmacology , Phosphoproteins/metabolism , Purines/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , Adaptor Proteins, Signal Transducing , Animals , Cell Cycle Proteins , Cell Line, Tumor , Class I Phosphatidylinositol 3-Kinases , Colonic Neoplasms/pathology , Eukaryotic Initiation Factors , Female , Humans , MAP Kinase Signaling System/drug effects , Mice , Mutation , Phosphatidylinositol 3-Kinases/genetics , Phosphorylation/drug effects , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , Ribosomal Protein S6/metabolism , TOR Serine-Threonine Kinases/metabolism , Xenograft Model Antitumor Assays , ras Proteins/genetics
2.
Leukemia ; 22(9): 1698-706, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18548104

ABSTRACT

The phosphatidylinositol 3-kinase (PI3K)/Akt and mammalian target of rapamycin complex 1 (mTORC1) signaling pathways are frequently activated in acute myelogenous leukemia (AML). mTORC1 inhibition with RAD001 induces PI3K/Akt activation and both pathways are activated independently, providing a rationale for dual inhibition of both pathways. PI-103 is a new potent PI3K/Akt and mTOR inhibitor. In human leukemic cell lines and in primary blast cells from AML patients, PI-103 inhibited constitutive and growth factor-induced PI3K/Akt and mTORC1 activation. PI-103 was essentially cytostatic for cell lines and induced cell cycle arrest in the G1 phase. In blast cells, PI-103 inhibited leukemic proliferation, the clonogenicity of leukemic progenitors and induced mitochondrial apoptosis, especially in the compartment containing leukemic stem cells. In contrast, apoptosis was not induced with RAD001 and IC87114 association, which specifically inhibits mTORC1 and p110delta activity, respectively. PI-103 had additive proapoptotic effects with etoposide in blast cells and in immature leukemic cells. Interestingly, PI-103 did not induce apoptosis in normal CD34(+) cells and had moderate effects on their clonogenic and proliferative properties. Here, we demonstrate that multitargeted therapy against PI3K/Akt and mTOR with PI-103 may be of therapeutic value in AML.


Subject(s)
Antineoplastic Agents/pharmacology , Furans/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Protein Kinases/drug effects , Pyridines/pharmacology , Pyrimidines/pharmacology , Apoptosis/drug effects , Bone Marrow Cells/drug effects , Bone Marrow Cells/pathology , Furans/therapeutic use , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Humans , Leukemia, Myeloid, Acute/pathology , Neoplastic Stem Cells/drug effects , Protein Kinase Inhibitors/therapeutic use , Pyridines/therapeutic use , Pyrimidines/therapeutic use , TOR Serine-Threonine Kinases , Tumor Cells, Cultured
3.
Biochem Soc Trans ; 35(Pt 2): 245-9, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17371250

ABSTRACT

PI3K (phosphoinositide 3-kinase) is a key regulator of cell growth, metabolism and survival. The frequent activation of the PI3K pathway in cancer has stimulated widespread interest in identifying potent and selective inhibitors of PI3K isoforms. The present paper highlights recent progress in identifying such molecules and the challenges that remain for efforts to pharmacologically target the PI3K family.


Subject(s)
Phosphatidylinositol 3-Kinases/metabolism , Animals , Cell Division , Cell Survival , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Neoplasms/enzymology , Phosphoinositide-3 Kinase Inhibitors
4.
Proc Natl Acad Sci U S A ; 101(50): 17456-61, 2004 Dec 14.
Article in English | MEDLINE | ID: mdl-15505216

ABSTRACT

Protein kinase inhibitors can be effective in treating selected cancers, but most suppress several kinases. Imatinib mesylate has been useful in the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia and B cell acute lymphoblastic leukemia through the inhibition of BCR-ABL tyrosine kinase activity. Imatinib mesylate has also been shown to inhibit KIT, ARG, and platelet-derived growth factor receptors alpha and beta, and potentially other tyrosine kinases. We have produced a mutant allele of BCR-ABL (T315A) that is uniquely inhibitable by the small molecule 4-amino-1-tert-butyl-3-(1-naphthyl)pyrazolo[3,4-d]pyrimidine and used it to demonstrate that sole suppression of BCR-ABL activity was insufficient to eliminate BCR-ABL(+) KIT(+)-expressing immature murine myeloid leukemic cells. In contrast, imatinib mesylate effectively eliminated BCR-ABL(+) KIT(+)-expressing leukemic cells. In the cellular context of mature myeloid cells and Pro/Pre B cells that do not express KIT, monospecific BCR-ABL inhibition was quantitatively as effective as imatinib mesylate in suppressing cell growth and inducing apoptosis. These results suggest that the therapeutic effectiveness of small molecule drugs such as imatinib mesylate could be due to the inhibitor's ability to suppress protein kinases in addition to the dominant target.


Subject(s)
Enzyme Inhibitors/pharmacology , Myeloproliferative Disorders/enzymology , Myeloproliferative Disorders/pathology , Phosphotyrosine , Protein-Tyrosine Kinases/antagonists & inhibitors , Apoptosis/drug effects , B-Lymphocytes/drug effects , B-Lymphocytes/pathology , Benzamides , Cell Cycle/drug effects , Cell Line , Cell Proliferation/drug effects , Drug Synergism , Fusion Proteins, bcr-abl , Gene Expression Regulation, Enzymologic , Humans , Imatinib Mesylate , Mutation/genetics , Myeloproliferative Disorders/genetics , Phosphotyrosine/metabolism , Piperazines/pharmacology , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-kit/metabolism , Pyrimidines/pharmacology , Tyrosine/genetics , Tyrosine/metabolism
5.
Annu Rev Cell Dev Biol ; 17: 405-33, 2001.
Article in English | MEDLINE | ID: mdl-11687495

ABSTRACT

A number of novel chemical methods for studying biological systems have recently been developed that provide a means of addressing biological questions not easily studied with other techniques. In this review, examples that highlight the development and use of such chemical approaches are discussed. Specifically, strategies for modulating protein activity or protein-protein interactions using small molecules are presented. In addition, methods for generating and utilizing novel biomolecules (proteins, oligonucleotides, oligosaccharides, and second messengers) are examined.


Subject(s)
Molecular Biology/methods , Oligonucleotides/chemical synthesis , Oligosaccharides/chemical synthesis , Protein Interaction Mapping/methods , Proteins/chemical synthesis , Animals , Combinatorial Chemistry Techniques , Forecasting , Humans , Models, Molecular , Oligonucleotides/chemistry , Oligonucleotides/metabolism , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Peptide Library , Protein Kinase Inhibitors , Proteins/genetics , Proteins/metabolism , Second Messenger Systems
6.
Proc Natl Acad Sci U S A ; 98(22): 12578-83, 2001 Oct 23.
Article in English | MEDLINE | ID: mdl-11675494

ABSTRACT

In addition to its well-established role in responding to phosphate starvation, the cyclin-dependent kinase Pho85 has been implicated in a number of other physiological responses of the budding yeast Saccharomyces cerevisiae, including synthesis of glycogen. To comprehensively characterize the range of Pho85-dependent gene expression, we used a chemical genetic approach that enabled us to control Pho85 kinase activity with a cell-permeable inhibitor and whole genome transcript profiling. We found significant phenotypic differences between the rapid loss of activity caused by inhibition and the deletion of the genomic copy of PHO85. We demonstrate that Pho85 controls the expression of not only previously identified glycogen synthetic genes, but also a significant regulon of genes involved in the cellular response to environmental stress. In addition, we show that the effects of this inhibitor are both rapid and reversible, making it well suited to the study of the behavior of dynamic signaling pathways.


Subject(s)
Cyclin-Dependent Kinases/physiology , Enzyme Inhibitors/pharmacology , Saccharomyces cerevisiae Proteins/physiology , Alleles , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/genetics , Environment , Gene Expression Regulation , Oligonucleotide Array Sequence Analysis , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Saccharomyces cerevisiae Proteins/genetics
7.
Curr Opin Chem Biol ; 5(4): 360-7, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11470597

ABSTRACT

New chemical methods that use small molecules to perturb cellular function in ways analogous to genetics have recently been developed. These approaches include both synthetic methods for discovering small molecules capable of acting like genetic mutations, and techniques that combine the advantages of genetics and chemistry to optimize the potency and specificity of small-molecule inhibitors. Both approaches have been used to study protein function in vivo and have provided insights into complex signaling cascades.


Subject(s)
Chemistry , Genetics , Signal Transduction , Chemical Phenomena , Mutation
8.
Trends Cell Biol ; 11(4): 167-72, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11306297

ABSTRACT

A chemical-genetic method for the generation of target-specific protein kinase inhibitors has been developed recently. This strategy utilizes a functionally silent active-site mutation to sensitize a target kinase to inhibition by a small molecule that does not inhibit wild-type kinases. Tyrosine and serine/threonine kinases are equally amenable to the drug-sensitization approach, which has been used to generate selective inhibitors of mutant Src-family kinases, Abl-family kinases, cyclin-dependent kinases, mitogen-activated kinases, p21-activated kinases and Ca(2+)/calmodulin-dependent kinases. The designed inhibitors are specific for the sensitized kinase in a cellular background where the wild-type kinase has been inactivated. By these means, kinase-sensitization has been used systematically to generate and analyze conditional alleles of several yeast protein kinases in vivo.


Subject(s)
Carrier Proteins/chemical synthesis , Intracellular Signaling Peptides and Proteins , Protein Kinases/genetics , Protein Kinases/therapeutic use , Animals , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Drug Delivery Systems/methods , Drug Delivery Systems/trends , Drug Design , Genetic Therapy/methods , Genetic Therapy/trends , Humans , Mutagenesis , Protein Kinases/drug effects
9.
Nat Cell Biol ; 3(3): 325-30, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11231586

ABSTRACT

Heterogeneous nuclear ribonucleoprotein K (hnRNP-K) is one of a family of 20 proteins that are involved in transcription and post-transcriptional messenger RNA metabolism. The mechanisms that underlie regulation of hnRNP-K activities remain largely unknown. Here we show that cytoplasmic accumulation of hnRNP-K is phosphorylation-dependent. Mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) efficiently phosphorylates hnRNP-K both in vitro and in vivo at serines 284 and 353. Serum stimulation or constitutive activation of ERK kinase (MEK1) results in phosphorylation and cytoplasmic accumulation of hnRNP-K. Mutation at ERK phosphoacceptor sites in hnRNP-K abolishes the ability to accumulate in the cytoplasm and renders the protein incapable of regulating translation of mRNAs that have a differentiation-control element (DICE) in the 3' untranslated region (UTR). Similarly, treatment with a pharmacological inhibitor of the ERK pathway abolishes cytoplasmic accumulation of hnRNP-K and attenuates inhibition of mRNA translation. Our results establish the role of MAPK/ERK in phosphorylation-dependent cellular localization of hnRNP-K, which is required for its ability to silence mRNA translation.


Subject(s)
Mitogen-Activated Protein Kinases/metabolism , Protein Biosynthesis , Ribonucleoproteins/metabolism , Arachidonate 15-Lipoxygenase/genetics , Blotting, Western , Cell Line , Cells, Cultured , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Genes, Reporter/genetics , Heterogeneous-Nuclear Ribonucleoprotein K , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Microscopy, Fluorescence , Mitogen-Activated Protein Kinases/genetics , Phosphorylation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Fusion Proteins/metabolism , Regulatory Sequences, Nucleic Acid/genetics , Transfection
10.
J Biol Chem ; 276(21): 18090-5, 2001 May 25.
Article in English | MEDLINE | ID: mdl-11259409

ABSTRACT

Modification of the ATP pocket on protein kinases allows selective use of an ATP analogue that exhibits high affinity for the altered kinases. Using this approach, we altered the ATP-binding site on JNK and identified N(6)-(2-phenythyl)-ATP, a modified form of ATP that exhibits high specificity and affinity for the modified, but not the wild type form, of JNK. Using modified JNK and its ATP analogue enables the detection of novel JNK substrates. Among substrates identified using this approach is heterogeneous nuclear ribonucleoprotein K, which is involved in transcription and post-transcriptional mRNA metabolism. The newly identified substrate can be phosphorylated by JNK on amino acids 216 and 353, which contribute to heterogeneous nuclear ribonucleoprotein K mediated transcriptional activities.


Subject(s)
JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinase Kinases/metabolism , Adenosine Triphosphate/genetics , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Cell Line , Humans , MAP Kinase Kinase 4 , Mitogen-Activated Protein Kinase Kinases/genetics , Molecular Sequence Data , Mutation , Phosphorylation , Substrate Specificity , Transcriptional Activation
11.
Biopolymers ; 60(3): 220-8, 2001.
Article in English | MEDLINE | ID: mdl-11774228

ABSTRACT

The problem of identifying downstream targets of kinase phosphorylation remains a challenge despite technological advances in genomics and proteomics. A recent approach involves the generation of kinase mutants that can uniquely use "orthogonal" ATP analogs to phosphorylate substrates in vivo. Using structure-based design, mutants of several protein kinase superfamily members have been found; robust and general methods are needed, however, for altering the nucleotide specificity of the remaining kinases in the genome. Here we demonstrate the application of a new phage display technique for direct functional selection to the identification of a tyrosine kinase mutant with the ability to use N6-benzyl-ATP. Our method produces, in five rounds of selection, a mutant identical to the best orthogonal Src kinase found to date. In addition, we isolate from a larger library of kinase mutants a promiscuous clone capable of using many different ATP analogs. This approach to engineering orthogonal kinases, combined with others, will facilitate the mapping of phosphorylation targets of any kinase in the genome.


Subject(s)
Peptide Library , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Arginine/chemistry , Base Sequence , Blotting, Western , Catalysis , DNA/metabolism , Escherichia coli/metabolism , Kinetics , Models, Biological , Models, Chemical , Models, Molecular , Molecular Sequence Data , Mutation , Phosphorylation , Protein Engineering , Sequence Homology, Amino Acid
12.
Biochemistry ; 39(47): 14400-8, 2000 Nov 28.
Article in English | MEDLINE | ID: mdl-11087392

ABSTRACT

Engineered protein kinases with unnatural nucleotide specificity and inhibitor sensitivity have been developed to trace kinase substrate targets. We first engineered unnatural nucleotide specificity into v-Src by mutating one residue, isoleucine 338, to alanine. This position is highly conserved among all kinases in the sense that it is always occupied by either a large hydrophobic residue or threonine. Because of the conservation of this residue and the highly conserved fold of the kinase family, we have attempted to generalize the engineering of all kinases on the basis of our success with v-Src. Although many kinases can be similarly engineered using v-Src as a blueprint, we encountered one kinase, c-Abl, which when mutated, does not display the ability to accept unnatural ATP analogues. To overcome this failure of the engineered c-Abl (T315A) to accept unnatural nucleotides, we developed a new strategy for introducing unnatural nucleotide specificity into kinases. We generated a chimeric kinase in which regions of the kinase domain of c-Abl were swapped with the corresponding regions of v-Src (I338A). Specifically, we engineered two chimeras in which the N-terminal lobe of the SH1 domain of c-Abl was swapped with that of v-Src. These kinase chimeras were found to have the same unnatural nucleotide specificity as that of v-Src (I338A), while retaining the peptide specificity of c-Abl. Thus, these chimeric kinases are suitable for identifying the direct substrates of c-Abl. These engineered chimeric enzymes provide a new strategy for constructing kinases with tailor-made ligand binding properties.


Subject(s)
Adenosine Triphosphate/genetics , Adenosine Triphosphate/metabolism , Enzyme Inhibitors/pharmacology , Oncogene Protein pp60(v-src)/genetics , Proto-Oncogene Proteins c-abl/genetics , Proto-Oncogene Proteins c-abl/metabolism , Recombinant Fusion Proteins/metabolism , src-Family Kinases/genetics , Adenosine Triphosphate/analogs & derivatives , Amino Acid Sequence , Animals , Binding Sites/genetics , Catalytic Domain/genetics , Consensus Sequence , Humans , Isoleucine/genetics , Mice , Molecular Sequence Data , Oncogene Protein pp60(v-src)/metabolism , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phosphopeptides/genetics , Phosphopeptides/metabolism , Protein Engineering , Proto-Oncogene Proteins c-abl/antagonists & inhibitors , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/chemical synthesis , Sequence Deletion , Substrate Specificity/genetics , src-Family Kinases/antagonists & inhibitors , src-Family Kinases/metabolism
13.
Nat Cell Biol ; 2(10): 677-85, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11025657

ABSTRACT

The p21-activated kinases (PAKs) are effectors for the Rho-family GTPase Cdc42p. Here we define the in vivo function of the kinase activity of the budding yeast PAK Cla4p, using cla4 alleles that are specifically inhibited by a cell-permeable compound that does not inhibit the wild-type kinase. CLA4 kinase inhibition in cells lacking the partially redundant PAK Ste20p causes reversible SWE1-dependent cell-cycle arrest and gives rise to narrow, highly elongated buds in which both actin and septin are tightly polarized to bud tips. Inhibition of Cla4p does not prevent polarization of F-actin, and cytokinesis is blocked only in cells that have not formed a bud before inhibitor treatment; cell polarization and bud emergence are not affected by Cla4p inhibition. Although localization of septin to bud necks is restored in swe1Delta cells, cytokinesis remains defective. Inhibition of Cla4p activity in swe1Delta cells causes a delay of bud emergence after cell polarization, indicating that this checkpoint may mediate an adaptive response that is capable of promoting budding when Cla4p function is reduced. Our data indicate that CLA4 PAK activity is required at an early stage of budding, after actin polarization and coincident with formation of the septin ring, for early bud morphogenesis and assembly of a cytokinesis site.


Subject(s)
Cyclins/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins , Saccharomycetales/cytology , Alleles , Amino Acid Sequence , Cell Compartmentation , Cell Cycle Proteins , Cell Division/genetics , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/genetics , Enzyme Activation , Fungal Proteins/isolation & purification , Intracellular Signaling Peptides and Proteins , MAP Kinase Kinase Kinases , Molecular Sequence Data , Mutagenesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Pyrazoles/pharmacology , Pyrimidines/pharmacology
14.
Nature ; 407(6802): 395-401, 2000 Sep 21.
Article in English | MEDLINE | ID: mdl-11014197

ABSTRACT

Protein kinases have proved to be largely resistant to the design of highly specific inhibitors, even with the aid of combinatorial chemistry. The lack of these reagents has complicated efforts to assign specific signalling roles to individual kinases. Here we describe a chemical genetic strategy for sensitizing protein kinases to cell-permeable molecules that do not inhibit wild-type kinases. From two inhibitor scaffolds, we have identified potent and selective inhibitors for sensitized kinases from five distinct subfamilies. Tyrosine and serine/threonine kinases are equally amenable to this approach. We have analysed a budding yeast strain carrying an inhibitor-sensitive form of the cyclin-dependent kinase Cdc28 (CDK1) in place of the wild-type protein. Specific inhibition of Cdc28 in vivo caused a pre-mitotic cell-cycle arrest that is distinct from the G1 arrest typically observed in temperature-sensitive cdc28 mutants. The mutation that confers inhibitor-sensitivity is easily identifiable from primary sequence alignments. Thus, this approach can be used to systematically generate conditional alleles of protein kinases, allowing for rapid functional characterization of members of this important gene family.


Subject(s)
Alleles , Enzyme Inhibitors/pharmacology , Protein Kinase Inhibitors , Protein Kinases/genetics , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , CDC28 Protein Kinase, S cerevisiae/antagonists & inhibitors , CDC28 Protein Kinase, S cerevisiae/genetics , Carbazoles/pharmacology , Cell Cycle , Fungal Proteins/antagonists & inhibitors , Gene Expression , Humans , Indole Alkaloids , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Molecular Sequence Data , Mutagenesis , Protein Structure, Tertiary , Proteins/pharmacology , Saccharomyces cerevisiae , Sequence Homology, Amino Acid , Transcription, Genetic
15.
Article in English | MEDLINE | ID: mdl-10940260

ABSTRACT

Small molecules that modulate the activity of biological signaling molecules can be powerful probes of signal transduction pathways. Highly specific molecules with high affinity are difficult to identify because of the conserved nature of many protein active sites. A newly developed approach to discovery of such small molecules that relies on protein engineering and chemical synthesis has yielded powerful tools for the study of a wide variety of proteins involved in signal transduction (G-proteins, protein kinases, 7-transmembrane receptors, nuclear hormone receptors, and others). Such chemical genetic tools combine the advantages of traditional genetics and the unparalleled temporal control over protein function afforded by small molecule inhibitors/activators that act at diffusion controlled rates with targets.


Subject(s)
Genetic Techniques , Ligands , Protein Engineering , Alleles , Amino Acid Sequence , Animals , Binding Sites , GTP Phosphohydrolases/chemistry , Humans , Kinesins/chemistry , Kinesins/genetics , Models, Biological , Models, Molecular , Molecular Sequence Data , Myosins/chemistry , Myosins/genetics , Protein Kinases/chemistry , Proteins/chemical synthesis , Signal Transduction
16.
J Exp Med ; 191(8): 1443-8, 2000 Apr 17.
Article in English | MEDLINE | ID: mdl-10770810

ABSTRACT

Signal transduction through the B cell antigen receptor (BCR) is altered in B cells that express a receptor that recognizes self-antigen. To understand the molecular basis for the change in signaling in autoreactive B cells, a transgenic model was used to isolate a homogeneous population of tolerant B lymphocytes. These cells were compared with a similar population of naive B lymphocytes. We show that the BCR from naive B cells enters a detergent-insoluble domain of the cell within 6 s after antigen binding, before a detectable increase in BCR phosphorylation. This fraction appears to be important for signaling because it is enriched for lyn kinase but lacks CD45 tyrosine phosphatase and because the BCR that moves into this domain becomes more highly phosphorylated. Partitioning of the BCR into this fraction is unaffected by src family kinase inhibition. Tolerant B cells do not efficiently partition the BCR into the detergent-insoluble domain, providing an explanation for their reduced tyrosine kinase activation and calcium flux in response to antigen. These results identify an early, regulated step in antigen receptor signaling and self-tolerance.


Subject(s)
B-Lymphocytes/immunology , Receptors, Antigen, B-Cell/metabolism , Self Tolerance , Animals , Autoimmunity , Chickens , Mice , Mice, Transgenic , Muramidase/immunology , Phosphorylation , Receptors, Antigen, B-Cell/genetics , Signal Transduction
18.
J Biol Chem ; 274(44): 31373-81, 1999 Oct 29.
Article in English | MEDLINE | ID: mdl-10531338

ABSTRACT

Distinguishing the cellular functions carried out by enzymes of highly similar structure would be simplified by the availability of isozyme-selective inhibitors. To determine roles played by individual members of the large myosin superfamily, we designed a mutation in myosin's nucleotide-binding pocket that permits binding of adenine nucleotides modified with bulky N(6) substituents. Introduction of this mutation, Y61G in rat myosin-Ibeta, did not alter the enzyme's affinity for ATP or actin and actually increased its ATPase activity and actin-translocation rate. We also synthesized several N(6)-modified ADP analogs that should bind to and inhibit mutant, but not wild-type, myosin molecules. Several of these N(6)-modified ADP analogs were more than 40-fold more potent at inhibiting ATP hydrolysis by Y61G than wild-type myosin-Ibeta; in doing so, these analogs locked Y61G myosin-Ibeta tightly to actin. N(6)-(2-methylbutyl) ADP abolished actin filament motility mediated by Y61G, but not wild-type, myosin-Ibeta. Furthermore, a small fraction of inhibited Y61G molecules was sufficient to block filament motility mediated by mixtures of wild-type and Y61G myosin-Ibeta. Introduction of Y61G myosin-Ibeta molecules into a cell should permit selective inhibition by N(6)-modified ADP analogs of cellular processes dependent on myosin-Ibeta.


Subject(s)
Adenosine Diphosphate/analogs & derivatives , Adenosine Triphosphatases/antagonists & inhibitors , Isoenzymes/antagonists & inhibitors , Myosins/antagonists & inhibitors , Actins/metabolism , Adenosine Diphosphate/pharmacology , Adenosine Triphosphatases/genetics , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Binding Sites , Chickens , Dictyostelium , Hydrolysis , Isoenzymes/genetics , Models, Molecular , Molecular Sequence Data , Movement , Mutation , Mutation, Missense , Myosins/genetics , Protein Binding , Protein Engineering , Rats
19.
Chem Biol ; 6(9): 671-8, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10467133

ABSTRACT

BACKGROUND: Small-molecule inhibitors that can target individual kinases are powerful tools for use in signal transduction research. It is difficult to find such compounds because of the enormous number of protein kinases and the highly conserved nature of their catalytic domains. Recently, a novel, potent, Src family selective tyrosine kinase inhibitor was reported (PP1). Here, we study the structural basis for this inhibitor's specificity for Src family kinases. RESULTS: A single residue corresponding to Ile338 (v-Src numbering; Thr338 in c-Src) in Src family tyrosine kinases largely controls PP1's ability to inhibit protein kinases. Mutation of Ile338 to a larger residue such as methionine or phenylalanine in v-Src makes this inhibitor less potent. Conversely, mutation of Ile338 to alanine or glycine increases PP1's potency. PP1 can inhibit Ser/Thr kinases if the residue corresponding to Ile338 in v-Src is mutated to glycine. We have accurately predicted several non-Src family kinases that are moderately (IC(50) approximately 1 microM) inhibited by PP1, including c-Abl and the MAP kinase p38. CONCLUSIONS: Our mutagenesis studies of the ATP-binding site in both tyrosine kinases and Ser/Thr kinases explain why PP1 is a specific inhibitor of Src family tyrosine kinases. Determination of the structural basis of inhibitor specificity will aid in the design of more potent and more selective protein kinase inhibitors. The ability to desensitize a particular kinase to PP1 inhibition of residue 338 or conversely to sensitize a kinase to PP1 inhibition by mutation should provide a useful basis for chemical genetic studies of kinase signal transduction.


Subject(s)
Enzyme Inhibitors/pharmacology , Mitogen-Activated Protein Kinases , Protein-Tyrosine Kinases/antagonists & inhibitors , Proteins/pharmacology , src-Family Kinases/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Enzyme Inhibitors/chemistry , Isoleucine/physiology , Models, Molecular , Molecular Sequence Data , Mutagenesis/physiology , Mutation , Protein Conformation , Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/genetics , Proteins/chemistry , Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , p38 Mitogen-Activated Protein Kinases , src-Family Kinases/chemistry , src-Family Kinases/genetics
20.
Pharmacol Ther ; 82(2-3): 337-46, 1999.
Article in English | MEDLINE | ID: mdl-10454210

ABSTRACT

Protein phosphorylation is the major post-translational modification used by eukaryotic cells to control cellular signaling. Protein kinases have emerged as attractive drug targets because heightened protein kinase activity has been associated with several proliferative diseases, most notably cancer and restenosis. Until now, it has been very difficult to confirm the utility of protein kinases as inhibitor targets because very few small molecules that selectively inhibit one particular kinase are known. Discovery of highly specific kinase inhibitors has been slow because the protein family contains approximately 2000 members, all of which share a conserved active site fold. Recent work in several laboratories has sought to circumvent the problem of kinase structural degeneracy by engineering drug sensitivity into Src family tyrosine kinases and mitogen-activated protein kinases through site-directed mutagenesis. By introducing a unique non-naturally occurring amino acid into a conserved region of the enzyme's binding site, a target protein kinase can be rapidly sensitized to a small molecule. Introduction of the engineered kinase into a cell line or animal model should greatly expedite the investigation of protein kinase inhibition as a viable drug treatment. The purpose of this review is to summarize these recent advances in protein kinase drug sensitization.


Subject(s)
Mitogen-Activated Protein Kinases/genetics , Protein Kinase Inhibitors , Protein Kinases/genetics , Proteins/chemical synthesis , src-Family Kinases/genetics , Animals , Drug Design , Fibroblasts/pathology , Forecasting , Humans , In Vitro Techniques , Protein-Tyrosine Kinases/genetics , Sensitivity and Specificity , Sequence Homology
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