Cytokinins, like other phytohormones, act in plants as signaling molecules at very low concentrations. The system that mediates between their chemical recognition and the responses that they induce requires a hormone receptor that, together with down-stream located elements, forms a signaling network, converting the signal into a specific response. Identification of the cytokinin-binding histidine kinases CRE1/AHK4, AHK3, and AHK2 as cytokinin receptors in Arabidopsis was an important milestone in the elucidation of cytokinin signal transduction pathways. Their molecular characterization through the use of transgenic E. coli strains revealed that a variety of cytokinin compounds may have signaling functions, but only with specific receptors. This indicates that differential ligand specificities of the receptors may be a mechanism to fine-tune the various cytokinin responses. This chapter describes the detailed protocol of a method employing transgenic E. coli which substantially contributes to our understanding of cytokinin perception, a crucial step in the cytokinin regulation of diverse plant growth and development processes.

• MeSH
• Arabidopsis enzymologie   MeSH
• beta-galaktosidasa genetika   metabolismus   MeSH
• biotest metody   MeSH
• cytokininy metabolismus   farmakologie   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• proteinkinasy genetika   metabolismus   MeSH
• regulace genové exprese u bakterií účinky léků   MeSH
• regulátory růstu rostlin metabolismus   farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cytokinins are a class of plant hormones that regulate the cell cycle and diverse developmental and physiological processes. Several compounds have been identified that antagonize the effects of cytokinins. Based on structural similarities and competitive inhibition, it has been assumed that these anticytokinins act through a common cellular target, namely the cytokinin receptor. Here, we examined directly the possibility that various representative classical anticytokinins inhibit the Arabidopsis cytokinin receptors CRE1/AHK4 (cytokinin response 1/Arabidopsis histidine kinase 4) and AHK3 (Arabidopsis histidine kinase 3). We show that pyrrolo[2,3-d]pyrimidine and pyrazolo[4,3-d]pyrimidine anticytokinins do not act as competitors of cytokinins at the receptor level. Flow cytometry and microscopic analyses revealed that anticytokinins inhibit the cell cycle and cause disorganization of the microtubular cytoskeleton and apoptosis. This is consistent with the hypothesis that they inhibit regulatory cyclin-dependent kinase (CDK) enzymes. Biochemical studies demonstrated inhibition by selected anti-cytokinins of both Arabidopsis and human CDKs. X-ray determination of the crystal structure of a human CDK2-anticytokinin complex demonstrated that the antagonist occupies the ATP-binding site of CDK2. Finally, treatment of human cancer cell lines with anticytokinins demonstrated their ability to kill human cells with similar effectiveness as known CDK inhibitors.

• MeSH
• apoptóza MeSH
• Arabidopsis metabolismus   MeSH
• buněčný cyklus MeSH
• chronická myeloidní leukemie metabolismus   patologie   MeSH
• cyklin-dependentní kinasa 2 antagonisté a inhibitory   metabolismus   MeSH
• cytokininy antagonisté a inhibitory   metabolismus   MeSH
• cytoskelet MeSH
• financování organizované MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• nádory kostí metabolismus   patologie   MeSH
• nádory prsu metabolismus   patologie   MeSH
• osteosarkom metabolismus   patologie   MeSH
• proliferace buněk účinky léků   MeSH
• proteinkinasy metabolismus   MeSH
• proteiny huseníčku metabolismus   MeSH
• průtoková cytometrie MeSH
• pyrimidiny farmakologie   MeSH
• receptory buněčného povrchu metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• transportní proteiny MeSH
• Check Tag
• lidé MeSH