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Title: Autophosphorylation is crucial for CDK-activating kinase (Ee;CDKF;1) activity and complex formation in leafy spurge

Author
item JIA, YING - TEXAS A&M UNIVERSITY
item Anderson, James
item Chao, Wun

Submitted to: Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/26/2010
Publication Date: 2/1/2011
Citation: Jia, Y., Anderson, J.V., Chao, W.S. 2011. Autophosphorylation is crucial for CDK-activating kinase (Ee;CDKF;1) activity and complex formation in leafy spurge. Plant Science. 180:259-267.

Interpretive Summary: Ee;CDKF;1 protein is involved in a phosphorylation cascade linked to early stages of cell cycle progression. 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 loss 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. We propose that CDKF;1 is at the top of their kinase signaling pathways. When CDKF;1s are synthesized in vivo, these molecules autophosphorylate and form homo protein complexes immediately. CDKF;1 complex is then able to phosphorylate and activate CDKs during cell cycle progression and to activate other CDK activating kinases as Shimotohno et al. have shown for At;CDKF;1.

Technical Abstract: Ee;CDKF;1 protein is involved in a phosphorylation cascade linked to early stages of cell cycle progression. 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 loss 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.