Several cell cycle events require specific forms of the cyclin-CDK complexes. It has been known for some time that cyclins not only contribute by activating the CDK but also by choosing substrates and/or specifying the location of the CDK holoenzyme. There are several examples of B-type cyclins identifying certain peptide motifs in their specific substrates through a conserved region in their structure. Such interactions were not known for the G1 class of cyclins, which are instrumental in helping the cell decide whether or not to commit to a new cell cycle, a function that is non-redundant with B-type cylins in budding yeast. In this dissertation, I have presented evidence that some G1 cyclins in budding yeast, Cln1/2, specifically identify substrates by interacting with a leucine-proline rich sequence different from the ones used by B-type cyclins. These “LP” type docking motifs determine cyclin specificity, promote phosphorylation of suboptimal CDK sites and multi-site phosphorylation of substrates both in vivo and in vitro. Subsequently, we have discovered the substrate-binding region in Cln2 and further showed that this region is highly conserved amongst a variety of fungal G1 cyclins from budding yeasts to molds and mushrooms, thus suggesting a conserved function across fungal evolution. Interestingly, this region is close to but not same as the one implicated in B-type cyclins to binding substrates. We discovered that the main effect of obliterating this interaction is to delay cell cycle entry in budding yeast, such that cells begin DNA replication and budding only at a larger than normal cell size, possibly resulting from incomplete multi-site phosphorylation of several key substrates. The docking-deficient Cln2 was also defective in promoting polarized bud morphogenesis. Quite interestingly, we found that a CDK inhibitor, Far1, could regulate the Cln2-CDK1 activity partly by inhibiting the Cln2-substrate interaction, thus demonstrating that docking interactions can be targets of regulation. Finally, by studying many fungal cyclins exogenously expressed in budding yeast, we discovered that some have the ability to make the CDK hyper-potent, which suggests that these cyclins confer special properties to the CDK. My work provides mechanistic clues for cyclinspecific events during the cell cycle, demonstrates the usefulness of synthetic strategies in problem solving and also possibly resolves long-standing uncertainties regarding functions of some cell cycle proteins.
| Identifer | oai:union.ndltd.org:umassmed.edu/oai:escholarship.umassmed.edu:gsbs_diss-1873 |
| Date | 21 October 2014 |
| Creators | Bhaduri, Samyabrata |
| Publisher | eScholarship@UMassChan |
| Source Sets | University of Massachusetts Medical School |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Morningside Graduate School of Biomedical Sciences Dissertations and Theses |
| Rights | Copyright is held by the author, with all rights reserved. |
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