ABSTRACT
In response to DNA damage and replication blocks, cells activate pathways that arrest the cell cycle and induce the transcription of genes that facilitate repair. In mammals, ATM (ataxia telangiectasia mutated) kinase together with other checkpoint kinases are important components in this response. We have cloned the rat and human homologs of Saccharomyces cerevisiae Rad 53 and Schizosaccharomyces pombe Cds1, called checkpoint kinase 2 (chk2). Complementation studies suggest that Chk2 can partially replace the function of the defective checkpoint kinase in the Cds1 deficient yeast strain. Chk2 was phosphorylated and activated in response to DNA damage in an ATM dependent manner. Its activation in response to replication blocks by hydroxyurea (HU) treatment, however, was independent of ATM. Using mass spectrometry, we found that, similar to Chk1, Chk2 can phosphorylate serine 216 in Cdc25C, a site known to be involved in negative regulation of Cdc25C. These results suggest that Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway. Activation of Chk2 might not only delay mitotic entry, but also increase the capacity of cultured cells to survive after treatment with gamma-radiation or with the topoisomerase-I inhibitor topotecan.
Subject(s)
DNA Damage , DNA Repair/genetics , Protein Kinases , Protein Serine-Threonine Kinases/physiology , Proteins/physiology , ras-GRF1 , Alkylating Agents/pharmacology , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Cycle/genetics , Cell Cycle Proteins/metabolism , Cells, Cultured , Checkpoint Kinase 2 , Cloning, Molecular , DNA, Complementary/genetics , DNA, Fungal/drug effects , DNA, Fungal/genetics , DNA, Fungal/radiation effects , DNA-Binding Proteins , Enzyme Inhibitors/pharmacology , Fungal Proteins/genetics , Fungal Proteins/metabolism , Fungal Proteins/physiology , Gamma Rays , Genetic Complementation Test , Humans , Hydroxyurea/pharmacology , Phosphorylation , Protein Processing, Post-Translational , Rats , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/radiation effects , Schizosaccharomyces/drug effects , Schizosaccharomyces/genetics , Schizosaccharomyces/radiation effects , Schizosaccharomyces pombe Proteins , Signal Transduction , Species Specificity , Topoisomerase I Inhibitors , Topotecan/pharmacology , Tumor Suppressor ProteinsABSTRACT
Eotaxin has been found to bind exclusively to a single chemokine receptor, CCR3. Using expression sequence tag screening of an activated monocyte library, a second chemokine has been identified; it was expressed and purified from a Drosophila cell culture system and appears to only activate CCR3. Eotaxin-2, MPIF-2, or CKbeta-6, is a human CC chemokine with low amino acid sequence identity to other chemokines. Eotaxin-2 promotes chemotaxis and Ca2+ mobilization in human eosinophils but not in neutrophils or monocytes. Cross-desensitization calcium mobilization experiments using purified eosinophils indicate that eotaxin and MCP-4, but not RANTES, MIP-1alpha, or MCP-3, can completely cross-desensitize the calcium response to eotaxin-2 on these cells, indicating that eotaxin-2 shares the same receptor used by eotaxin and MCP-4. Eotaxin-2 was the most potent eosinophil chemoattractant of all the chemokines tested. Eotaxin-2 also displaced 125I-eotaxin bound to the cloned CCR3 stably expressed in CHO cells (CHO-CCR3) and to freshly isolated human eosinophils with affinities similar to eotaxin and MCP-4. 125I-Eotaxin-2 binds with high affinity to eosinophils and both eotaxin and cold eotaxin-2 displace the ligand with equal affinity. Eotaxin and eotaxin-2 promote a Ca2+ transient in RBL-2H3 cells stably transfected with CCR3 (RBL-2H3-CCR3) and both ligands cross-desensitized the response of the other but not the response to LTD4. The data indicate that eotaxin-2 is a potent eosinophil chemotactic chemokine exerting its activity solely through the CCR3 receptor.
