title: A short motif in Arabidopsis CDK inhibitor ICK1 decreases the protein level, probably through a ubiquitin-independent mechanism.
abstract: The ICK/KRP family of cyclin-dependent kinase (CDK) inhibitors modulates the activity of plant CDKs through protein binding. Previous work has shown that changing the levels of ICK/KRP proteins by overexpression or downregulation affects cell proliferation and plant growth, and also that the ubiquitin proteasome system is involved in degradation of ICK/KRPs. We show in this study that the region encompassing amino acids 21 to 40 is critical for ICK1 levels in both Arabidopsis and yeast. To determine how degradation of ICK1 is controlled, we analyzed the accumulation of hemagglutinin (HA) epitope-tagged ICK1 proteins in yeast mutants defective for two ubiquitin E3 ligases. The highest level of HA-ICK1 protein was observed when both the N-terminal 1-40 sequence was removed and the SCF (SKP1-Cullin1-F-box complex) function disrupted, suggesting the involvement of both SCF-dependent and SCF-independent mechanisms in the degradation of ICK1 in yeast. A short motif consisting of residues 21-30 is sufficient to render green fluorescent protein (GFP) unstable in plants and had a similar effect in plants regardless of whether it was fused to the N-terminus or C-terminus of GFP. Furthermore, results from a yeast ubiquitin receptor mutant rpn10Δ indicate that protein ubiquitination is not critical in the degradation of GFP-ICK1(1-40) in yeast. These results thus identify a protein-destabilizing sequence motif that does not contain a typical ubiquitination residue, suggesting that it probably functions through an SCF-independent mechanism.
authors.affiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, SK, S7N 5E5, Canada.
authors.forename: Qin
authors.initials: Q
authors.lastname: Li
authors.affiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, SK, S7N 5E5, Canada.
authors.forename: Xianzong
authors.initials: X
authors.lastname: Shi
authors.affiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, SK, S7N 5E5, Canada.
authors.forename: Shengjian
authors.initials: S
authors.lastname: Ye
authors.affiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, SK, S7N 5E5, Canada.
authors.forename: Sheng
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, SK, S7N 5E5, Canada.
authors.forename: Ron
authors.initials: R
authors.lastname: Chan
authors.affiliation: Department of Anatomy and Cell Biology, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada.
authors.forename: Troy
authors.initials: T
authors.lastname: Harkness
authors.affiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, SK, S7N 5E5, Canada. hong.wang@usask.ca.
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
mesh_terms: D020816:Amino Acid Motifs; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D044842:Cullin Proteins / Q000235:genetics; D050756:Cyclin-Dependent Kinase Inhibitor Proteins / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D049452:Green Fluorescent Proteins / Q000235:genetics; D007976:Leupeptins / Q000494:pharmacology; D009154:Mutation; D030821:Plants, Genetically Modified; D055550:Protein Stability; D013696:Temperature; D025801:Ubiquitin / Q000378:metabolism; D015003:Yeasts / Q000187:drug effects / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; C100318:Cullin 1; D044842:Cullin Proteins; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; C114853:ICK1 protein, Arabidopsis; D007976:Leupeptins; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde
keywords: Arabidopsis thaliana; CDK inhibitor; ICK/KRP; cell cycle; protein stability; ubiquitin proteasome system