Materials and methods br Results br Discussion The
Materials and methods
Discussion The BV-tag-based electrochemiluminescence immunoassay is an extremely sensitive assay. It has been employed for detecting analytes present in only picogram quantities (e.g., bacterial toxins , Milrinone ), measuring the activity of basic carboxypeptidase , and monitoring Stat6/P578 protein–peptide interaction . However, to our knowledge, it has not been used for measuring kinase activity. The BV ECL ACK1 assay described here is sensitive enough to detect as little as 8 nM (240 fmol/well) of the kinase product. This exceptional sensitivity enabled the determination of Ki values in the picomolar range while avoiding problems from bottoming out of the assay. Furthermore, in this assay, the reaction/detection mix is washed away inside the reader. This results in a very low CV (∼5%), yet the washing step eliminates any potential interference from color or fluorescent compounds. This assay is generally applicable to any kinase for which a phospho-specific antibody recognizing the kinase product is available. Labeling such an antibody with BV-tag N-hydroxysuccinimide ester is a reliable and straightforward procedure. Several assay formats are commonly used for the measurement of protein kinase activity. These can be broadly divided into three categories: (i) radioactive formats using filter separation, SPA, or flash plates; (ii) nonradioactive and non-antibody-based assay formats such as Z′-LYTE (Invitrogen) and IQ Kinase Assay (Pierce); and (iii) nonradioactive antibody-based assays such as HTRF (Cisbio), LANCE (PerkinElmer), DELFIA, and ELISA (for reviews, see Refs. , ). The disadvantage of filtration, flash plate, and SPA assays is associated radioisotope use in that each requires either [γ-32P]ATP or, more commonly, [γ-33P]ATP. Another disadvantage of these formats is the inherent limitations on their use with high ATP concentrations. The specific activity of radioisotope tracers is limited by high ATP concentrations with consequent loss of sensitivity as ATP concentrations are increased.
Plant growth and development is controlled via an intricate network of extracellular and intracellular signaling pathways, which involve both negative, and positive regulatory signals , , , . Regulation of the cell cycle during developmental processes involves differential expression of some cell-cycle genes in specific tissues in response to plant growth regulators . Cell division is controlled by the activity of cyclin dependent kinases (CDKs), which are regulated by the presence of cyclins , . CDKs associate with specific cyclins for activation and the timing of CDK activation depends on the kinetics and localization of cyclin expression , , . CDK complexes composed of cyclin and catalytic CDK subunits are activated in a periodic manner to promote cell-cycle transitions or arrest the cell cycle at different phases , , . Morgan has demonstrated that CDKs have been implicated in the control of gene transcription in plants. CDK activity may be regulated at various levels, such as gene expression, phosphorylation, differential subcellular localization, and interaction with regulatory proteins. Plant CDK inhibitors such as ICK1 and ICK2 show distinct distributions in different tissues and have specific roles in development , , . The cellular events associated with leaf development and the down-regulation of homobox genes involved in the formation of leaf have been reported , , . A knowledge of the timing, sequence, and localization of CDK activation and inactivation is key for understanding developmental processes. In this report, we have identified a cyclin D1 interacting protein (p22) that can inhibit the kinase activity of CDK and reverse the phenotypic effects of cyclin D1 overexpression, suggesting that p22 and cyclin Dl act in concert to control the early stage of leaf formation. Materials and methods