ABSTRACT
Topoisomerase (topo) IIα and IIß maintain genome stability and are targets for anti-tumor drugs. In this study, we demonstrate that the decatenation checkpoint is regulated, not only by topo IIα, as previously reported, but also by topo IIß. The decatenation checkpoint is most efficient when both isoforms are present. Regulation of this checkpoint and sensitivity to topo II-targeted drugs is influenced by the C-terminal domain (CTD) of the topo II isoforms and by a conserved non-catalytic tyrosine, Y640 in topo IIα and Y656 in topo IIß. Deletion of most of the CTD of topo IIα, while preserving the nuclear localization signal (NLS), enhances the decatenation checkpoint and sensitivity to topo II-targeted drugs. In contrast, deletion of most of the CTD of topo IIß, while preserving the NLS, and mutation of Y640 in topo IIα and Y656 in topo IIß inhibits these activities. Structural studies suggest that the differential impact of the CTD on topo IIα and topo IIß function may be due to differences in CTD charge distribution and differential alignment of the CTD with reference to transport DNA. Together these results suggest that topo IIα and topo IIß cooperate to maintain genome stability, which may be distinctly modulated by their CTDs.
Subject(s)
Antigens, Neoplasm/chemistry , Cell Cycle Checkpoints/physiology , Chromosomal Instability/physiology , DNA Topoisomerases, Type II/chemistry , DNA-Binding Proteins/chemistry , Amino Acid Sequence , Animals , Antigens, Neoplasm/drug effects , Antigens, Neoplasm/genetics , Antigens, Neoplasm/physiology , Cell Line , DNA Damage , DNA Topoisomerases, Type II/drug effects , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/physiology , DNA, Complementary/genetics , DNA-Binding Proteins/drug effects , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Drug Resistance, Neoplasm , Fibroblasts , HL-60 Cells , Humans , Mice , Mutagenesis, Site-Directed , Protein Domains , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Structure-Activity Relationship , Topoisomerase II Inhibitors/pharmacologyABSTRACT
Plants and animals have evolved structurally related innate immune sensors, designated NLRs, to detect intracellular nonself molecules. NLRs are modular, consisting of N-terminal coiled-coil (CC) or TOLL/interleukin-1 receptor (TIR) domains, a central nucleotide-binding (NB) domain, and C-terminal leucine-rich repeats (LRRs). The polymorphic barley mildew A (MLA) locus encodes CC-containing allelic immune receptors recognizing effectors of the pathogenic powdery mildew fungus. We report the crystal structure of an MLA receptor's invariant CC domain, which reveals a rod-shaped homodimer. MLA receptors also self-associate in vivo, but self-association appears to be independent of effector-triggered receptor activation. MLA CC mutants that fail to self-interact impair in planta cell death activity triggered by the CC domain alone and by an autoactive full-length MLA receptor that mimics its ATP-bound state. Thus, CC domain-dependent dimerization of the immune sensor defines a minimal functional unit and implies a role for the dimeric CC module in downstream immune signaling.
Subject(s)
Hordeum/immunology , Plant Proteins/chemistry , Receptors, Immunologic/chemistry , Amino Acid Sequence , Ascomycota , Cell Death , Chromatography, Gel , Crystallography, X-Ray , Genes, Reporter , Genetic Loci , Hordeum/cytology , Hordeum/genetics , Hordeum/microbiology , Hydrophobic and Hydrophilic Interactions , Molecular Sequence Data , Mutagenesis, Site-Directed , Plant Leaves/cytology , Plant Leaves/genetics , Plant Leaves/immunology , Plant Proteins/genetics , Plant Proteins/immunology , Plant Proteins/metabolism , Plants, Genetically Modified , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Secondary , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Two-Hybrid System TechniquesABSTRACT
Topoisomerase (topo) II catalyzes topological changes in DNA. Although both human isozymes, topo IIα and ß are phosphorylated, site-specific phosphorylation of topo IIß is poorly characterized. Using LC-MS/MS analysis of topo IIß, cleaved with trypsin, Arg C or cyanogen bromide (CNBr) plus trypsin, we detected four +80-Da modified sites: tyr656, ser1395, thr1426 and ser1545. Phosphorylation at ser1395, thr1426 and ser1545 was established based on neutral loss of H(3) PO(4) (-98 Da) in the CID spectra and on differences in 2-D-phosphopeptide maps of (32) P-labeled wild-type (WT) and S1395A or T1426A/S1545A mutant topo IIß. However, phosphorylation at tyr656 could not be verified by 2-D-phosphopeptide mapping of (32) P-labeled WT and Y656F mutant protein or by Western blotting with phosphotyrosine-specific antibodies. Since the +80-Da modification on tyr656 was observed exclusively during cleavage with CNBr and trypsin, this modification likely represented bromination, which occurred during CNBr cleavage. Re-evaluation of the CID spectra identified +78/+80-Da fragment ions in CID spectra of two peptides containing tyr656 and tyr711, confirming bromination. Interestingly, mutation of only tyr656, but not ser1395, thr1326 or ser1545, decreased topo IIß activity, suggesting a functional role for tyr656. These results, while identifying an important tyrosine in topo IIß, underscore the importance of careful interpretation of modifications having the same nominal mass.