Studies of arabidopsis cyclin-dependent kinase inhibitors : protein-protein interactions, phosphorylation and stability

The cyclin-dependent kinase (CDK) inhibitors have been demonstrated to be an important component in the regulation of plant cell cycle. Although they share a conserved CDK inhibitory region with a family of CDK inhibitors in mammals, the plant CDK inhibitors are very different from the animal and yeast CDK inhibitors. Thus studies of the plant CDK inhibitors could provide insight on the molecular mechanisms regulating the cell cycle in plants as well as the differences between plants and animals. The research described in this thesis investigated the seven Arabidopsis CDK inhibitors ICKs in terms of transgenic expression, phosphorylation, stability and interactions with other proteins. ICKs were expressed in transgenic Arabidopsis plants as fusion proteins with the green fluorescent protein (GFP). Consistent with the previous studies on ICK1, ICK2 and ICK4, overexpression of all seven ICKs inhibited plant growth and resulted in plants with serrated leaves and flowers with altered morphology. A Survey based on large a number of independent transformants showed that GFP-ICK3 and GFP-ICK4 had weaker phenotypic effects compared to other GFP-ICKs. The Western blotting results showed that all GFP-ICKs were expressed at a low level in general. The levels of GFP-ICK3 and GFP-ICK4 were the lowest, suggesting that the weaker effects for ICK3 and ICK4 may partly be due to low protein levels. Treatments with MG132, an inhibitor of the proteasome, resulted in moderate but clear accumulation of fusion proteins for ICK1, ICK5, ICK6 and ICK7 in plants, suggesting that the proteasome is involved in the degradation of these proteins. To study the state of protein phosphorylation, the proteins extracted from the plants were treated with calf intestinal phosphatase (CIP). The CIP treatment caused a faster migration of the GFP fusion protein for ICK1, ICK2, ICK5, ICK6 and ICK7, while the effect was not observed for control GFP and other non-specific proteins, indicating that these proteins can be phosphorylated in plants. The shift also differed among ICKs. Interestingly, dephosphorylation of ICK7 might have rendered it less stable. The protein pulldown experiments using p13Suc1-conjugated agarose beads showed that GFP-ICK4, GFP-ICK5 and GFP-ICK6 could associate with the CDK complex, similar to what has been shown for ICK1 and ICK2. CIP treatments of the p13Suc1 affinity-purified proteins also showed that ICK1, ICK2, ICK5 and ICK6 associated with the CDK complex were phosphorylated. Attempts were also made to isolate peptide aptamers that are able to interact with ICKs for the purpose for expressing such an aptamer in plants. However, an aptamer that has a strong ability to interact with ICKs in two of yeast two-hybrid systems was not identified. In addition, the analysis of Arabidopsis CYCD3;1 for its interaction with ICK1 using a series of deletion mutants showed that the removal of both the N-terminal and C-terminal regions of CYCD3;1 greatly reduced or abolished the interaction with ICK1. In summary, transgenic Arabidopsis plants have been obtained for expressing each of the seven Arabidopsis CDK inhibitors fused to GFP. The results confirmed and extended previous finding that overexpression of a CDK inhibitor inhibits plant growth as well as changes plant morphology. The observation that the ICK fusion proteins were generally at low and often undetectable levels, in comparison to much higher levels of the GFP protein, suggests that ICKs are unstable in the cell. Results from the MG132 experiments indicate that the 26S proteasome may play a role in the degradation of ICK1, ICK5, ICK6 and ICK7. Results from CIP treatments further show that most ICKs, particularly ICK1, ICK2, ICK5, ICK6 and ICK7, can be phosphorylated in vivo. Interestingly, ICK7 stability may depend on the status of protein phosphorylation. This study provides new understanding on how the family of proteins is regulated at the post-transcriptional level and the differences among Arabidopsis CDK inhibitors