THE ROLE OF N-TERMINAL REGIONS IN REGULATING THE LEVEL OF PLANT ICK CYCLIN-DEPENDENT KINASE INHIBITORS

Plants have a family of cyclin-dependent kinase (CDK) inhibitors named ICKs (interactor/inhibitor of CDK), which are important cell cycle regulators and can modulate CDK activity through direct binding. The model plant Arabidopsis thaliana has seven members in the ICK family. Previous results show that changing the levels of ICKs by either overexpression or down-regulation affects plant growth and plant morphology, and the effects depend on ICK expression levels, indicating that the function of ICKs is dosage-dependent. Thus, it is important to understand how the ICK levels are regulated. Several independent studies indicated that the ubiquitin proteasome system is involved in the degradation of ICKs. However, the functional sequence signals in ICKs for regulating their levels remain largely unknown. In this study, to identify sequences involved in regulating the levels of ICKs, the N-terminal regions of ICKs were fused to the green fluorescence protein (GFP) and expressed in Arabidopsis. Results showed that the presence of these N-terminal regions can dramatically reduce the GFP protein level. Since previous work showed that a motif as short as 10 amino acid residues could greatly affect the level of GFP fusion protein, 10-residue fragments of ICK4 and ICK6 were analyzed by fusing to GFP. Results showed that ICK666-75 led to a much reduced level of the fusion protein. Interestingly, this fragment belongs to a conserved motif in ICKs and the corresponding sequence of ICK1 has been shown to dramatically reduce GFP fusion protein expression. Mapping ICK2, ICK3, ICK4 and ICK6 using fragments about 20 residues in length identified several other sequences that could reduce the GFP expression level in Arabidopsis. Since all the ICK3 fragments were capable of dramatically decreasing the level of a reporter GFP protein, they were further analyzed in yeast and E. coli. ICK338-60 and ICK361-83 could reduce the level of GFP in both yeast and E. coli, implying a ubiquitin-independent mechanism. ICK321-37 could reduce the GFP level only in yeast suggesting that it functions differently from the other two sequences. Furthermore, ICK321-37 belongs to another conserved motif among ICKs. Results from this study provide new understanding regarding the role of N-terminal regions in regulating the level of ICKs. They also raise new questions for future investigation on this family of plant cell cycle regulators