Classical Anticytokinins Do Not Interact with Cytokinin Receptors but Inhibit Cyclin-dependent Kinases

Abstract

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. 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. Cytokinins are plant hormones that play essential roles in the regulation of various aspects of plant growth and development (1Mok D-W. Mok M.-C. Annu. Rev. Plant Physiol. Plant Mol. Biol. a of with of structural of in and that are known as cytokinins are that to the of their as are of the plant cell and the of cell is class of plant of is and in cell Cytokinins have been to and of kinase as as the of of the physiological and developmental that are by as the and of the of and Mok Mok M.-C. and Plant Plant Plant are at in to cell cycle cytokinin receptors identified in Arabidopsis Plant Physiol. Plant Physiol. Plant Physiol. and Plant Physiol. cytokinin receptors have been identified in Arabidopsis histidine kinase Arabidopsis histidine kinase cytokinin response 1/Arabidopsis histidine kinase cyclin-dependent Arabidopsis histidine kinase Arabidopsis histidine kinase cytokinin response 1/Arabidopsis histidine kinase cyclin-dependent and are histidine with a and kinase and has been that the cytokinin is by a through a complex of the that has been known in and the is in by Plant Biol. Plant Biol. Plant Plant development of and of a physiological is in studies of of cytokinin has been on the that cytokinins to cellular receptor and it to compounds that have cytokinin structural to the cytokinins to to cytokinin receptor the of cytokinins. cytokinin as the of the the with that cytokinin in Annu. Rev. Plant Physiol. a of and and their ability to inhibit in various and a of identified as anticytokinins by of their structural to cytokinins and their effects by the cytokinin it that these compounds through with a common cellular target, the cytokinin receptor Mok Mok M.-C. and that cytokinin receptors are the of cytokinin receptors been in of cytokinin to of we show that representative anticytokinins are not competitive of Arabidopsis cytokinin the as a representative we show that anticytokinins inhibit cell cycle and cause cellular consistent with to known CDK inhibitors. We CDK inhibition by anticytokinins in and and the of to the ATP-binding of human CDK2. of anticytokinins in human cancer cells and to and the as of the and Plant Physiol. Plant Physiol. cytokinin as in Plant Physiol. of and on and Plant Physiol. 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Mok M.-C. Annu. Rev. Plant Physiol. Plant Mol. Biol. a of with of structural of in and that are known as cytokinins are that to the of their as cytokinins. Cytokinins are of the plant cell and the of cell is class of plant of is and in cell Cytokinins have been to and of kinase as as the of of the physiological and developmental that are by as the and of the of and Mok Mok M.-C. and Plant Plant Plant are at in to cell cycle cytokinin receptors identified in Arabidopsis Plant Physiol. Plant Physiol. Plant Physiol. and Plant Physiol. cytokinin receptors have been identified in Arabidopsis histidine kinase Arabidopsis histidine kinase cytokinin response 1/Arabidopsis histidine kinase cyclin-dependent Arabidopsis histidine kinase Arabidopsis histidine kinase cytokinin response 1/Arabidopsis histidine kinase cyclin-dependent and are histidine with a and kinase and has been that the cytokinin is by a through a complex of the that has been known in and the is in by Plant Biol. Plant Biol. 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Plant Physiol. 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We human plant structural is structure has been human is to Arabidopsis of the that the ATP-binding of and in and are structure of and in that the occupies the ATP-binding of the structural of and to that the structure of occupies the as the in the with the of is similar to CDK as and of the inhibition and studies to the of anticytokinins and the CDK and on the of human and plant the of of cells of the cancer cell to and the CDK and a not of on the growth of cancer cell and with the of not in plant cells with and Arabidopsis on a with of to growth of both of in growth by by and in the Arabidopsis growth the of the have the of of compounds that been as cytokinin and to act as competitive at the cytokinin receptor studies selected anticytokinins and of the identified Arabidopsis cytokinin receptors and revealed that these compounds the receptors in a inhibit the of cytokinin with the receptors in these do not the possibility that the anticytokinins inhibit the known Arabidopsis cytokinin not in possibility is to anticytokinins not cytokinin of the response in Arabidopsis of show growth of the receptors Plant Plant inhibition of to have of anticytokinins on cytokinin of the of their of similarities plant cells and cells with the CDK and Plant compounds cell cycle at the and in We demonstrated that and inhibit the of Arabidopsis and in to studies that to human and occupies the as the structure of the kinase in a with the representative anticytokinins cell not in plant of and the of is in various human cancer cell of cytokinin revealed that processes. the inhibition of the of at the of and of the by treatment on and that have are in with the of and Plant Physiol. demonstrated that in both and cell cells are to the effects of is of that the anticytokinins a similar in both plant and human cycle inhibition and of are of cancer CDK inhibition of the compounds in are with of the cell cycle of is the treatment of cancer on and CDK inhibition structural have effects on the of as it to structural in to effectiveness in human is on the of human and ability to growth of human cancer cells is similar cellular of and namely the of by that by and in a in plant and these effects on cell cycle the ability of and to and of the of by anticytokinins and of these that the of anticytokinins is not directly to cytokinin at known their with cytokinins to a with their effects on the cell in they to cell cycle to inhibit and cell of cytokinin we that it is to the classical of of at the compounds and that anticytokinins inhibit human and We have the of of compounds that been as cytokinin and to act as competitive at the cytokinin receptor studies selected anticytokinins and of the identified Arabidopsis cytokinin receptors and revealed that these compounds the receptors in a inhibit the of cytokinin with the receptors in these do not the possibility that the anticytokinins inhibit the known Arabidopsis cytokinin not in possibility is to anticytokinins not cytokinin of the response in Arabidopsis of show growth of the receptors Plant Plant inhibition of to have of anticytokinins on cytokinin of the of their of similarities plant cells and cells with the CDK and Plant compounds cell cycle at the and in We demonstrated that and inhibit the of Arabidopsis and in to studies that to human and occupies the as the structure of the kinase in a with the representative anticytokinins cell not in plant of and the of is in various human cancer cell of cytokinin revealed that processes. the inhibition of the of at the of and of the by treatment on and that have are in with the of and Plant Physiol. demonstrated that in both and cell cells are to the effects of is of that the anticytokinins a similar in both plant and human cycle inhibition and of are of cancer CDK inhibition of the compounds in are with of the cell cycle of is the treatment of cancer on and CDK inhibition structural have effects on the of as it to structural in to effectiveness in human is on the of human and ability to growth of human cancer cells is similar cellular of and namely the of by that by and in a in plant and these effects on cell cycle the ability of and to and of the of by anticytokinins and of these that the of anticytokinins is not directly to cytokinin at known their with cytokinins to a with their effects on the cell in they to cell cycle to inhibit and cell of cytokinin we that it is to the classical of of at the compounds and that anticytokinins inhibit human and We are to of Plant the with of and We and with of cytometry and and of the with with