Submitted to: Plant Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/24/2006
Publication Date: 2/28/2007
Citation: Chao, W.S., Serpe, M.D., Jia, Y., Shelver, W.L., Anderson, J.V., Umeda, M. 2007. Potential roles for autophosphorylation, kinase activity, and abundance of a CDK-activating kinase (Ea;CDKF;1) during growth in leafy spurge. Plant Molecular Biology. 63(3):365-379. DOI:10.1007/s11103-006-9094-y. Interpretive Summary: Leafy spurge is a deep rooted perennial weed that propagates both by seeds and underground adventitious buds located on the crown and roots (crown and root buds). As buds develop during the normal growing season, they are maintained in a quiescent state. Existing evidence indicates that growth-arrest may be a result of interactions between signaling pathways controlling dormancy and those controlling the cell cycle. To enhance our understanding of growth and development during seed germination and vegetative propagation, a cell cycle gene encoding CDK-activating kinase (CAK1Ee) was cloned from leafy spurge. The CAK1Ee protein is involved in a phosphorylation cascade linked to early stages of cell cycle progression. The function of CAK1Ee gene has been confirmed based on its ability to rescue a yeast temperature sensitive CAK mutant and in vitro kinase assays. Site-directed mutagenesis of CAK1Ee indicated that two threonine residues (Thr291 and Thr296) were mutually responsible for autophosphorylation and for phosphorylating its substrate protein, cyclin-dependent kinase (CDK). Also, autophosphorylation was an intra-molecular activity. CAK1Ee polyclonal and phosphoprotein-specific antibodies were used to examine CAK phosphorylation in relation to seed germination and bud growth. Phosphorylated CAK was lower in dry and imbibed seeds but increased during seed germination. During crown bud development; however, we observed greater levels of phosphorylated CAK in small buds compared to that of larger buds. Moreover, Thr-291 remains phosphorylated in vivo at all times, which may be an important phenomenon for those early regulatory enzymes in cell cycle signaling pathways.
Technical Abstract: Leafy spurge (Euphorbia esula L.) is a deep rooted perennial weed that propagates both by seeds and underground adventitious buds located on the crown and roots. To enhance our understanding of growth and development during seed germination and vegetative propagation, a leafy spurge gene (accession number AF230740) encoding a CDK-activating kinase (Ee;CDKF;1) involved in cell cycle progression was identified and its function was confirmed based on its ability to rescue a yeast temperature sensitive CAK mutant (GF2351) and through in vitro kinase assays. Site-directed mutagenesis of Ee;CDKF;1 indicated that two threonine residues (Thr291 and Thr296) were mutually responsible for intra-molecular autophosphorylation and for phosphorylating its substrate protein, cyclin-dependent kinase (CDK). Polyclonal antibodies generated against the Ee;CDKF;1 protein or against a phosphorylated Ee;CDKF;1 peptide [NERYGSL(pT)SC] were used to examine abundance and phosphorylation of CDKF; 1 during seed germination and bud growth. The levels of CDKF;1 were lower in dry or imbibed seeds than in germinating seeds or seedlings. Differences in CDKF;1 were also observed during adventitious bud development; small buds appeared to have greater levels of CDKF;1 than large buds. Similar patterns of CDKF;1 expression were detected with either the polyclonal antibody developed using the CDKF;1 protein or the phosphorylated peptide. These results indicated that Thr291 is constitutively phosphorylated in vivo and associated with Ee;CDKF;1 activity. Our results further suggest that a certain level of CDKF;1 activity is maintained in most tissues and may be an important phenomenon for enzymes that regulate early steps in cell cycle signaling pathways. Abbreviations: CDK, cyclin-dependent kinase; CAK, CDK-activating kinase; CTD, C-terminal domain of the largest subunit of RNA polymerase II ; MBP, maltose binding protein; GST, glutathione S-transferase