Potential roles for autophosphorylation, kinase activity, and abundance of a CDK-activating kinase (Ee;CDKF;1) during growth in leafy spurge

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 No. 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.

A CDK-related kinase regulates the length and assembly of flagella in Chlamydomonas

“…Most CDKs are phosphorylated by other kinases, and a few can phosphorylate themselves (Chao et al, 2007). For LF2p, there are at least three isoforms.…”

The homoeologous genes for the Rec8-like meiotic cohesin in wheat: structure, function, and evolutionary implication

“…The amplicon was cloned into two expression plasmid vectors pGEX-4T-1 (Amersham Biosciences, Piscataway, NJ, USA) and pMAL-c2X (New England Biolabs, Ipswich, MA, USA), leading to two distinct constructs ( pGEX-R26 and pMAL-R26 ). After verified by sequencing, these two constructs were transformed into E. coli strain BL21-Star (DE3) (Invitrogen Corporation, Grand Island, NY, USA) for fusion protein induction as described by Chao et al (2007). Upon IPTG (isopropyl β-D-1-thiogalactopyranoside) induction, the fusion polypeptides, pGEX-R26 and pMAL-R26, were accumulated as insoluble pellets and resolubilized after sonication.…”

“…The anti-pGEX-R26 crude serum was affinity-purified as described by Chao et al (2007) with minor modifications. The affinity-purified pMAL-R26 polypeptide was first coupled to AminoLink coupling resin using the AminoLink Plus Immobilization Kit (Pierce Biotechnology, Rockford, IL, USA) and then incubated with the crude serum.…”

“…Protein extraction, immunoprecipitation, and Western blotting were performed following the procedures of Chao et al (2007). About 400 mg anthers at each of the four distinct meiotic stages (interphase, early prophase I, metaphase I/anaphase I, and metaphase II/anaphase II) were collected for protein extraction in the Western blotting analysis.…”

Changes in the expression of carbohydrate metabolism genes during three phases of bud dormancy in leafy spurge

“…For antibody purification, crude antiserum from two rabbits was combined and passed through a column containing immobilized P1 then a column containing immobilized P2 to obtain P1 and P2 antibodies. Using these antibodies, immunoprecipitation was carried out according to Chao et al (2007). For immunoblot analysis, protein samples were phenol extracted as described by Wang et al (1992).…”