phosphothreonine and rottlerin

Macrophage-derived chemokine [CC chemokine ligand 22 (CCL22)] and thymus- and activation-regulated chemokine (CCL17) mediate cellular effects, principally by binding to their receptor CC chemokine receptor 4 (CCR4) and together, constitute a multifunctional chemokine/receptor system with homeostatic and inflammatory roles within the body. This study demonstrates that CCL22 and CCL17 stimulate pertussis toxin-sensitive elevation of intracellular calcium in the CEM leukemic T cell line and human peripheral blood-derived T helper type 2 (Th2) cells. Inhibition of phospholipase C (PLC) resulted in the abrogation of chemokine-mediated calcium mobilization. Chemokine-stimulated calcium responses were also abrogated completely by the inhibition of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor-mediated calcium release. Chemotactic responses of CEM and human Th2 cells to CCL17 and CCL22 were similarly abrogated by inhibition of PLC and inhibition of novel, Ca2+-independent/diacylglycerol-dependent protein kinase C (PKC) isoforms. Inhibition of Ins(1,4,5)P3 receptor-mediated calcium release from intracellular stores had no effect on chemotactic responses to CCR4 ligands. Taken together, this study provides compelling evidence of an important role for PLC and diacylglycerol-dependent effector mechanisms (most likely involving novel PKC isoforms) in CCL17- and CCL22-stimulated, directional cell migration. In this regard, CCL22 stimulates phosphatidylinositol-3 kinase-independent phosphorylation of the novel delta isoform of PKC at threonine 505, situated within its activation loop--an event closely associated with increased catalytic activity.

Topics: Acetophenones; Benzopyrans; Calcium; Calcium Channels; Calcium Signaling; Catalytic Domain; Cell Line, Tumor; Chemokine CCL17; Chemokine CCL22; Chemokines, CC; Chemotaxis; Chromones; Diglycerides; Estrenes; Humans; Indoles; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Leukemia-Lymphoma, Adult T-Cell; Morpholines; Pertussis Toxin; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Phosphothreonine; Protein Processing, Post-Translational; Pyrroles; Pyrrolidinones; Receptors, CCR4; Receptors, Chemokine; Receptors, Cytoplasmic and Nuclear; Recombinant Fusion Proteins; T-Lymphocytes; Th2 Cells

The serine/threonine protein kinase family is a large and diverse group of enzymes that are involved in the regulation of multiple cellular pathways. Elevated kinase activity has been implicated in many diseases and frequently targeted for the development of pharmacological inhibitors. Therefore, non-radioactive antibody-based kinase assays that allow high throughput screening of compound libraries have been developed. However, they require a generation of antibodies against the phosphorylated form of a specific substrate. We report here a time-resolved fluorescence assay platform that utilizes a commercially-available generic anti-phospho-threonine antibody and permits assaying kinases that are able to phosporylate threonin residues on protein substrates. Using this approach, we developed an assay for Cdc7/Dbf4 kinase activity, determined the K(m) for ATP, and identified rottlerin as a non-ATP competitive inhibitor of this enzyme.

Topics: Acetophenones; Adenosine Triphosphate; Antibodies; Benzopyrans; Cell Cycle Proteins; Enzyme Inhibitors; Fluorescent Dyes; Fluoroimmunoassay; Humans; Kinetics; Minichromosome Maintenance Complex Component 2; Nuclear Proteins; Phosphorylation; Phosphothreonine; Protein Serine-Threonine Kinases; Saccharomyces cerevisiae Proteins; Sensitivity and Specificity