ARS | Publication request: Autophosphorylation affects protein complex formation and activity of CDK-activating kinase (Ee;CDKF;1) in leafy spurge (Euphorbia esula)

Submitted to: Weed Science Society of America Meeting Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: January 9, 2009
Publication Date: February 1, 2009
Citation: Chao, W.S., Jia, Y., Anderson, J.V. Autophosphorylation Affects Protein Complex Formation and Activity of CDK-Activating Kinase (Ee;CDKF;1) in Leafy Spurge (Euphorbia esula)[Abstract]. Weed Science Society of America Meeting Abstracts. Presentation #77.

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 (Ee;CDKF;1) was cloned from leafy spurge. The Ee;CDKF;1 protein is involved in a phosphorylation cascade linked to early stages of cell cycle progression. Two threonine residues (Thr291 and Thr296) are mutually responsible for autophosphorylation and for phosphorylating its substrate protein, cyclin-dependent kinase (CDK). Also, Thr291 remains phosphorylated in-vivo at all times. Yeast two-hybrid screening performed using Ee;CDKF;1 as a bait indicated that one of the interacting proteins was Ee;CDKF;1. Protein-protein interaction of Ee;CDKF;1 was further confirmed by yeast two-hybrid interaction and in-vitro pull-down assays. The full-length Ee;CDKF;1 was shown to interact with N-terminal, middle, and C-terminal regions of Ee;CDKF;1. Gel exclusion chromatography and/or native PAGE showed that Ee;CDKF;1 and GST-Ee;CDKF;1 were capable of forming both homo-dimer and -trimer complexes in its native state. In addition, both forms were autophosphorylated and able to phosphorylate CDK. Moreover, mutant forms of Ee;CDKF;1 (106G/A, 166K/A), which lost autophosphorylation capability completely, were unable to form homo protein complexes in their native state. The result thus demonstrated that autophosphorylation of Ee;CDKF;1 is crucial for both kinase activity and complex formation. Key words: Autophosphorylation, CDK-Activating Kinase, Ee;CDKF;1, Leafy Spurge.