ABSTRACT
Mixed-lineage leukemia fusion proteins activate their target genes predominantly by stimulating transcriptional elongation. A core component necessary for this activity is cyclin-dependent kinase 9. Here we explored the effectiveness of small molecules targeting this enzyme as potential therapeutics. A screen of seven compounds with anti-CDK9 activity applied to a panel of leukemia cell lines identified flavopiridol and the experimental inhibitor PC585 as superior in efficacy with inhibitory concentrations in the submicromolar range. Both substances induced rapid dephosphorylation of the RNA polymerase II C-terminal domain, accompanied by downregulation of CDK9-dependent transcripts for MYC and HOXA9. Global gene expression analysis indicated the induction of a general stress response program, culminating in widespread apoptosis. Importantly, colony-forming activity in leukemia lines and primary patient samples could be completely inhibited under conditions that did not affect native precursors from bone marrow. In vivo application in a mouse transplant model significantly delayed disease with PC585 showing also oral activity. These results suggest CDK9 inhibition as novel treatment option for mixed-lineage leukemia.
Subject(s)
Antineoplastic Agents/pharmacology , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Cyclin-Dependent Kinase 9/metabolism , Leukemia, Biphenotypic, Acute/drug therapy , Leukemia, Biphenotypic, Acute/metabolism , Protein Kinase Inhibitors/pharmacology , Animals , Antineoplastic Agents/administration & dosage , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/genetics , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Gene Expression Profiling , Gene Expression Regulation, Leukemic/drug effects , Humans , Leukemia, Biphenotypic, Acute/genetics , Mice , Phosphorylation , Protein Kinase Inhibitors/administration & dosage , Proto-Oncogene Proteins c-myc/genetics , RNA Polymerase II/metabolism , Tumor Stem Cell Assay , Xenograft Model Antitumor AssaysABSTRACT
Linkage analyses have implicated chromosome 7p21.3 as a susceptibility region for inflammatory bowel disease (IBD). Recently, the mouse phenotype with diarrhea and goblet cell dysfunction caused by anterior gradient protein 2 dysfunction was reported (European patent WO2004056858). The genes encoding for the human homologues AGR2 and AGR3 are localized on chromosome 7p21.3. The gene structures were verified and mutation detection was performed in 47 IBD patients. A total of 30 single nucleotide polymorphisms (SNPs) were tested for association to ulcerative colitis (UC, N = 317) and Crohn's disease (CD, N = 631) in a German cohort and verified in a UK cohort of 384 CD and 311 UC patients. An association signal was identified in the 5' region of the AGR2 gene (most significant SNP hcv1702494, nominal P(TDT) = 0.011, P(case/control) = 0.0007, OR = 1.34, combined cohort). The risk haplotype carried an odds ratio of 1.43 in the German population (P = 0.002). AGR2 was downregulated in UC patients as compared to normal controls (P < 0.001) and a trend toward lower expression was seen in carriers of the risk alleles. Luciferase assays of the AGR2 promoter showed regulation by the goblet cell-specific transcription factors FOXA1 and FOXA2. In summary, AGR2 represents an interesting new avenue into the etiopathophysiology of IBD and the maintenance of epithelial integrity.
Subject(s)
Carrier Proteins/genetics , Genetic Predisposition to Disease , Inflammatory Bowel Diseases/genetics , Neoplasm Proteins/genetics , Proteins/genetics , Carrier Proteins/biosynthesis , Cell Line , Colitis, Ulcerative/genetics , Colitis, Ulcerative/metabolism , Colitis, Ulcerative/physiopathology , Crohn Disease/genetics , Crohn Disease/metabolism , Crohn Disease/physiopathology , Hepatocyte Nuclear Factor 3-alpha/physiology , Hepatocyte Nuclear Factor 3-beta/physiology , Humans , Inflammatory Bowel Diseases/metabolism , Inflammatory Bowel Diseases/physiopathology , Mucoproteins , Neoplasm Proteins/biosynthesis , Oncogene Proteins , Proteins/metabolismABSTRACT
The beta(2) integrin LFA-1 is an important cell-cell adhesion receptor of the immune system. Evidence suggests that the molecule also participates in signaling and co-stimulatory function. We show here that clustering of the intracellular domain of the beta(2) chain but not of the alpha(L)- or beta(1)-cytoplasmic domains, respectively, triggers intracellular Ca(2+) mobilization in Jurkat cells. A beta(2)-specific NPXF motif, located in the C-terminal portion of the beta(2) tail, is required for Ca(2+) signaling, and we show that this motif is important for the induction of allo-specific target cell lysis by cytotoxic T cells in vitro. Significantly, the Ca(2+)-signaling capacity of the beta(2) integrin is abrogated in T cells that do not express the T cell receptor but may be reconstituted by co-expression of the T cell receptor-zeta chain. Our data suggest a specific function of the cytoplasmic domain of the beta(2) integrin chain in T cell signaling.
Subject(s)
CD18 Antigens/chemistry , CD18 Antigens/metabolism , Calcium/metabolism , Cytoplasm/metabolism , Lymphocyte Function-Associated Antigen-1/chemistry , Lymphocyte Function-Associated Antigen-1/metabolism , Signal Transduction , Amino Acid Motifs , Amino Acid Sequence , Blotting, Western , DNA/metabolism , Flow Cytometry , Humans , Jurkat Cells , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding , Protein Structure, Tertiary , Receptors, Antigen, T-Cell/metabolism , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , T-Lymphocytes/metabolism , Time Factors , TransfectionABSTRACT
CD4 recruitment to T cell receptor (TCR)-peptide-major histocompatibility class II complexes is required for stabilization of low affinity antigen recognition by T lymphocytes. The cytoplasmic portion of CD4 is thought to amplify TCR-initiated signal transduction via its association with the protein tyrosine kinase p56(lck). Here we describe a novel functional determinant in the cytosolic tail of CD4 that inhibits TCR-induced T cell activation. Deletion of two conserved hydrophobic amino acids from the CD4 carboxyl terminus resulted in a pronounced enhancement of CD4-mediated T cell costimulation. This effect was observed in the presence or absence of p56(lck), implying involvement of alternative cytosolic ligands of CD4. A two-hybrid screen with the intracellular portion of CD4 identified a previously unknown 33-kDa protein, ACP33 (acidic cluster protein 33), as a novel intracellular binding partner of CD4. Since interaction with ACP33 is abolished by deletion of the hydrophobic CD4 C-terminal amino acids mediating repression of T cell activation, we propose that ACP33 modulates the stimulatory activity of CD4. Furthermore, we demonstrate that interaction with CD4 is mediated by the noncatalytic alpha/beta hydrolase fold domain of ACP33. This suggests a previously unrecognized function for alpha/beta hydrolase fold domains as a peptide binding module mediating protein-protein interactions.
Subject(s)
CD4 Antigens/metabolism , Carrier Proteins/genetics , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , COS Cells , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cell Line , Cloning, Molecular , Endosomes/metabolism , Golgi Apparatus/metabolism , Humans , Ligands , Lymphocyte Activation , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Molecular Sequence Data , Mutation , Phenotype , Protein Binding , Sequence Homology, Amino Acid , Subcellular Fractions/metabolism , T-Lymphocytes/immunologyABSTRACT
Intracellular signaling pathways, which regulate the interactions of integrins with their ligands, affect a wide variety of biological functions. Here we provide evidence of how cytohesin-1, an integrin-binding protein and guanine-nucleotide exchange factor (GEF) for ARF GTPases, regulates cell adhesion. Mutational analyses of the beta-2 cytoplasmic domain revealed that the adhesive function of LFA-1 depends on its interaction with cytohesin-1, unless the integrin is activated by exogenous divalent cations. Secondly, cytohesin-1 induces expression of an extracellular activation epitope of LFA-1, and the exchange factor function is not essential for this activity. In contrast, LFA-1-mediated cell adhesion and spreading on intercellular cell adhesion molecule 1 is strongly inhibited by a cytohesin-1 mutant, which fails to catalyze ARF GDP-GTP exchange in vitro. Thus, cytohesin-1 is involved in the activation of LFA-1, most probably through direct interaction with the integrin, and induces cell spreading by its ARF-GEF activity. We therefore propose that both direct regulation of the integrin and concomitant changes in the membrane topology of adherent T cells are modulated by dissectable functions of cytohesin-1.
Subject(s)
ADP-Ribosylation Factors/physiology , CD18 Antigens/physiology , Cell Adhesion Molecules/physiology , Cell Adhesion/physiology , Lymphocyte Function-Associated Antigen-1/physiology , T-Lymphocytes/cytology , Animals , Cell Adhesion Molecules/genetics , Cell Size , Epitopes/chemistry , Guanine Nucleotide Exchange Factors , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , HeLa Cells , Humans , Intercellular Adhesion Molecule-1/physiology , Macromolecular Substances , Mice , Models, Molecular , Rats , Recombinant Fusion Proteins/physiology , Two-Hybrid System TechniquesABSTRACT
Recruitment of intracellular proteins to the plasma membrane is a commonly found requirement for the initiation of signal transduction events. The recently discovered pleckstrin homology (PH) domain, a structurally conserved element found in approximately 100 signaling proteins, has been implicated in this function, because some PH domains have been described to be involved in plasma membrane association. Furthermore, several PH domains bind to the phosphoinositides phosphatidylinositol-(4,5)-bisphosphate and phosphatidylinositol-(3,4,5)-trisphosphate in vitro, however, mostly with low affinity. It is unclear how such weak interactions can be responsible for observed membrane binding in vivo as well as the resulting biological phenomena. Here, we investigate the structural and functional requirements for membrane association of cytohesin-1, a recently discovered regulatory protein of T cell adhesion. We demonstrate that both the PH domain and the adjacent carboxyl-terminal polybasic sequence of cytohesin-1 (c domain) are necessary for plasma membrane association and biological function, namely interference with Jurkat cell adhesion to intercellular adhesion molecule 1. Biosensor measurements revealed that phosphatidylinositol-(3,4,5)-trisphosphate binds to the PH domain and c domain together with high affinity (100 nM), whereas the isolated PH domain has a substantially lower affinity (2-3 microM). The cooperativity of both elements appears specific, because a chimeric protein, consisting of the c domain of cytohesin-1 and the PH domain of the beta-adrenergic receptor kinase does not associate with membranes, nor does it inhibit adhesion. Moreover, replacement of the c domain of cytohesin-1 with a palmitoylation-isoprenylation motif partially restored the biological function, but the specific targeting to the plasma membrane was not retained. Thus we conclude that two elements of cytohesin-1, the PH domain and the c domain, are required and sufficient for membrane association. This appears to be a common mechanism for plasma membrane targeting of PH domains, because we observed a similar functional cooperativity of the PH domain of Bruton's tyrosine kinase with the adjacent Bruton's tyrosine kinase motif, a novel zinc-containing fold.
Subject(s)
Blood Proteins/chemistry , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/physiology , Cell Adhesion , Cell Membrane/metabolism , Phosphoproteins , src Homology Domains , Amino Acid Sequence , Animals , Biosensing Techniques , COS Cells , Cell Adhesion Molecules/biosynthesis , Cell Line , Conserved Sequence , Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP-Dependent Protein Kinases/metabolism , Glutathione Transferase/biosynthesis , Guanine Nucleotide Exchange Factors , Humans , Jurkat Cells , Molecular Sequence Data , Phosphatidylinositol Phosphates/metabolism , Polymerase Chain Reaction , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Sequence Alignment , T-Lymphocytes/physiology , Transfection , beta-Adrenergic Receptor KinasesABSTRACT
The Syk family tyrosine kinases play a crucial role in antigen receptor-mediated signal transduction, but their regulation and cellular targets remain incompletely defined. Following receptor engagement, phosphorylation of tyrosine residues within ZAP-70 and Syk is thought to control both kinase activity and recruitment of modulatory factors. We report here the characterization of novel mutants of ZAP-70 and Syk, in which conserved C-terminal tyrosine residues have been replaced by phenylalanines (ZAP YF-C, Syk YF-C). Both mutant kinases display a prominent gain-of-function phenotype in Jurkat T cells, as demonstrated by lymphokine promoter activation, tyrosine phosphorylation of potential targets in vivo, and elevated intracellular calcium mobilization. While the presence of p56-Lck was required for ZAP YF-C-induced signaling, Syk YF-C showed enhanced functional activity in Lck-deficient JCaM1 Jurkat cells. Our results implicate the C terminus of Syk family kinases as an important regulatory region modulating T cell activation.
Subject(s)
Enzyme Precursors/genetics , Lymphocyte Activation , Protein-Tyrosine Kinases/genetics , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/immunology , Amino Acid Sequence , Calcium/metabolism , Catalysis , Enzyme Precursors/metabolism , Humans , Interleukin-2/genetics , Intracellular Signaling Peptides and Proteins , Jurkat Cells , Molecular Sequence Data , Mutation , Promoter Regions, Genetic , Protein-Tyrosine Kinases/metabolism , Receptors, Antigen, B-Cell/metabolism , Receptors, Antigen, T-Cell/metabolism , Recombinant Fusion Proteins/metabolism , Signal Transduction , Syk Kinase , T-Lymphocytes/enzymology , ZAP-70 Protein-Tyrosine KinaseABSTRACT
Signal transduction through phosphoinositide 3-OH kinase (PI 3-kinase) has been implicated in the regulation of lymphocyte adhesion mediated by integrin receptors. Cellular phosphorylation products of PI 3-kinases interact with a subset of pleckstrin homology (PH) domains, a module that has been shown to recruit proteins to cellular membranes. We have recently identified cytohesin-1, a cytoplasmic regulator of beta2 integrin adhesion to intercellular adhesion molecule 1. We describe here that expression of a constitutively active PI 3-kinase is sufficient for the activation of Jurkat cell adhesion to intercellular adhesion molecule 1, and for enhanced membrane association of cytohesin-1. Up-regulation of cell adhesion by PI 3-kinase and membrane association of endogenous cytohesin-1 is abrogated by overexpression of the isolated cytohesin-1 PH domain, but not by a mutant of the PH domain which fails to associate with the plasma membrane. The PH domain of Bruton's tyrosine kinase (Btk), although strongly associated with the plasma membrane, had no effect on either membrane recruitment of cytohesin-1 or on induction of adhesion by PI 3-kinase. Having delineated the critical steps of the beta2 integrin activation pathway by biochemical and functional analyses, we conclude that PI 3-kinase activates inside-out signaling of beta2 integrins at least partially through cytohesin-1.
Subject(s)
CD18 Antigens/metabolism , Cell Adhesion Molecules/metabolism , Phosphatidylinositol 3-Kinases/physiology , Agammaglobulinaemia Tyrosine Kinase , Cell Adhesion/physiology , Cell Membrane/metabolism , Enzyme Activation/physiology , Fluorescent Antibody Technique , Gene Expression Regulation, Enzymologic/genetics , Guanine Nucleotide Exchange Factors , Humans , Immunoglobulin G/genetics , Intercellular Adhesion Molecule-1/metabolism , Jurkat Cells , Protein-Tyrosine Kinases/physiology , Recombinant Fusion Proteins/genetics , Signal Transduction/physiology , Up-Regulation/physiologySubject(s)
Integrins/metabolism , Signal Transduction , Amino Acid Sequence , Animals , Binding Sites , Cell Adhesion , Cytoplasm/metabolism , Cytoskeleton , Humans , Molecular Sequence DataABSTRACT
The avidity of integrin adhesion receptors for extracellular ligands is subject to dynamic regulation by intracellular programs that have yet to be elucidated. We describe here a protein, cytohesin-1, which specifically interacts with the intracellular portion of the integrin beta 2 chain (CD18). The molecule shows homology to the yeast SEC7 gene product and bears a pleckstrin homology (PH) domain. Overexpression of either the full-length cytohesin-1 or the SEC7 domain induces beta 2 integrin-dependent binding of Jurkat cells to ICAM-1, whereas expression of the isolated cytohesin-1 PH domain inhibits T cell receptor-stimulated adhesion. Similar inhibition is not exhibited by PH domains taken from other proteins, showing that the interaction is specific and that individual PH domains are capable of discriminating between alternative targets.