title: Autophosphorylation is crucial for CDK-activating kinase (Ee;CDKF;1) activity and complex formation in leafy spurge.
abstract: Ee;CDKF;1 protein is a leafy spurge (Euphorbia esula) CDK-activating kinase that 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. Gel exclusion chromatography and/or native PAGE showed that GST-CDKF;1, MBP-CDKF;1, GST-CDKF;1 devoid of GST, and endogenous Ee;CDKF;1 were capable of forming homo protein complexes which are in dimer, trimer, and/or higher molecular-mass complex in its native state. In addition, Ee;CDKF;1 complexes 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.
authors.affiliation: Department of Plant Science, North Dakota State University, Fargo, ND 58105, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Jia
authors.forename: James V
authors.initials: JV
authors.lastname: Anderson
authors.forename: Wun S
authors.initials: WS
authors.lastname: Chao
mesh_terms: D018844:Cyclin-Dependent Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D028481:Euphorbia / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics; D017343:Genes, Plant; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D020798:Two-Hybrid System Techniques; D000095043:Cyclin-Dependent Kinase-Activating Kinase
chemical_list: D011993:Recombinant Fusion Proteins; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase