The plant cyclin‐dependent kinase inhibitor ICK1 has distinct functional domains for in vivo kinase inhibition, protein instability and nuclear localization

Abstract

SummaryInteractor/inhibitor 1 of Cdc2 kinase (ICK1) from Arabidopsis thaliana is the ®rst plant cyclin-dependent kinase (CDK) inhibitor, and overexpression of ICK1 inhibits CDK activity, cell division and plant growth in transgenic plants. In this study, ICK1 and deletion mutants were expressed either alone or as green¯uor-escent protein (GFP) fusion proteins in transgenic Arabidopsis plants. Deletion of the C-terminal 15 or 29 amino acids greatly reduced or completely abolished the effects of ICK1 on the transgenic plants, and recombinant proteins lacking the C-terminal residues lost the ability to bind to CDK complex and the kinase inhibition activity, demonstrating the role of the conserved C-terminal domain in in vivo kinase inhibition. In contrast, the mutant ICK1 DN108 with the N-terminal 108 residues deleted had much stronger effects on plants than the full-length ICK1. Analyses demonstrated that this effect was not because of an enhanced ability of ICK1 DN108 protein to inhibit CDK activity, but a result of a much higher level of ICK1 DN108 protein in the plants, indicating that the N-terminal domain contains a sequence or element increasing protein instability in vivo. Furthermore, GFP-ICK1 protein was restricted to the nuclei in roots of transgenic plants, even with the C-terminal or the N-terminal domain deleted, suggesting that a sequence in the central domain of ICK1 is responsible for nuclear localization. These results provide mechanistic understanding about the function and regulation of this cell cycle regulator in plants.

Novel Functions of Plant Cyclin-Dependent Kinase Inhibitors, ICK1/KRP1, Can Act Non-Cell-Autonomously and Inhibit Entry into Mitosis

“…While this work was in progress, a similar intracellular localization of ICK1/KRP1 was reported by analyzing GFP fusions with ICK1/KRP1 (Zhou et al, 2003). Consistent with the report by Zhou and colleagues, we found that YFP fusions with the truncated ICK1/KRP1 109 localized to the nucleus and the cytoplasm in trichomes (Figures 3C, 3G, and 3K; data not shown); a cytoplasmic localization was even more prominent for the GUS:YFP:ICK1/KRP1 109 fusion protein ( Figures 3D, 3H, and 3L).…”

“…The limitation in detection of the full-length CKI argues that this domain might regulate the stability of ICK1/KRP1 protein. Consistently, Zhou et al (2003) recently reported that an N-terminally truncated version was present in much higher abundance than the full-length inhibitor.…”

“…For the fulllength version, we could not detect a band, although on RNA level the construct appeared even to be slightly stronger expressed than the truncated version (see Supplemental Figures 1A and 1B online). We and others have previously shown that a negative regulatory signal resides in the N terminus of the ICK1/KRP1 protein Zhou et al, 2003). The limitation in detection of the full-length CKI argues that this domain might regulate the stability of ICK1/KRP1 protein.…”

CULLIN 4-RING FINGER-LIGASE plays a key role in the control of endoreplication cycles in Arabidopsis trichomes

“…This gave rise to the possibility that one of the major targets of a CRL-complex in trichomes could be CDK inhibitors. We and others have previously shown that ICK1/KRP1 is an unstable protein and degraded by the proteasome pathway (32,(37)(38)(39).…”

Targeted Degradation of the Cyclin-Dependent Kinase Inhibitor ICK4/KRP6 by RING-Type E3 Ligases Is Essential for Mitotic Cell Cycle Progression duringArabidopsisGametogenesis

“…On the basis of sequence homology to p27 Kip1 , seven putative CDK inhibitors have been identified in Arabidopsis thaliana, designated as Interactors of Cdc2 Kinase (ICKs) or Kip-related proteins (KRPs). ICK1/KRP1 and ICK2/KRP2 in Arabidopsis were shown to be regulated through the action of the 26S proteasome (Zhou et al, 2003b;Weinl et al, 2005;Jakoby et al, 2006). Although it was recently reported that KRP1 degradation was dependent both on SCF SKP2b and the RING protein RKP (Ren et al, 2008), the physiological roles of these ICK/KRPs and the ubiquitin E3 ligases responsible for their degradation are largely unknown.…”