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Title: AUTOPHOSPHORYLATION AND KINASE ACTIVITY OF A CDK-ACTIVATING KINASE (CAK1EE) FROM LEAFY SPURGE (EUPHORBIA ESULA)

Author
item Chao, Wun
item SERPE, MARCELO - BOISE STATE UNIVERSITY
item JIA, YING - NDSU DEPT PLANT SCI
item Shelver, Weilin
item Anderson, James
item UMEDA, MASAAKI - THE UNIVERSITY OF TOKYO

Submitted to: American Society of Plant Biologists Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 8/5/2006
Publication Date: 8/5/2006
Citation: Chao, W.S., Serpe, M.D., Jia, Y., Shelver, W.L., Anderson, J.V., Umeda, M. Autophosphorylation and kinase activity of a cdk-activating kinase (cak1ee) from leafy spurge (Euphorbia esula). 2006 American Society of Plant Biologists Annual Meeting Abstracts. Available: http://abstracts.aspb.org/pb2006/public/P21/P21007.html

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 (Thr-291 and Thr-296) 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 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 through correlative inhibition. 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 (GF2351) and in vitro kinase assays. Site-directed mutagenesis of CAK1Ee indicated that two threonine residues (Thr-291 and Thr-296) 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. Key words: Leafy Spurge, CDK-Activating Kinase, autophosphorylation.