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Biochemistry of Cell Cycle Regulation

The long range goal of our lab is to understand biochemically how cell growth and division are regulated by checkpoints within the cell and by controls imposed from the surrounding tissues. The cyclin-dependent protein kinases (CDKs), whose activities are required for cell cycle transitions, are regulated via multiple mechanisms including specific association with a cyclin, multiple phosphorylations (both positive- and negative-acting), sensing of a threshold, and at least two feedback loops that combine to produce the precise and abrupt activation of protein kinase activity necessary for accurate cell cycle transitions. We study the protein kinases, phosphatases, and regulatory proteins that control CDKs. We also study the ubiquitin-mediated degradation of cyclins and other mitotic proteins and how this degradation is blocked by the Spindle-Assembly Checkpoint.

Eukaryotic Cell Cycle at a Biochemical Level

We seek to understand the eukaryotic cell cycle at a biochemical level. To get there, we use a variety of approaches including genetics and "pseudo genetics" in yeast, biochemistry and enzyme assays using cell extracts and purified proteins from yeast and mammalian cells, and structural analyses (in collaboration). Progressing from genetic identification of components through their study in vitro we hope to achieve a more complete understanding of these macroscopic cellular behaviors.

Speedy/Ringo Family of CDK Activators

The main focus of our studies on CDK activation concerns the Speedy/Ringo family of CDK activators. Though these proteins bear no sequence similarity to cyclins, they can activate CDKs, even in the absence of activating phosphorylation of the CDK, which is required for activation by cyclins. We have identified a family of at least four Ringo proteins with distinct expression patterns. Biochemical analyses of Ringo-CDK complexes suggest that they may jump start cell cycle transitions. Thus, a low amount of Ringo-CDK2 may activate CDC25 protein phosphatases, which in turn can activate the bulk of cyclin-CDK2 complexes and induce entry into S phase. We are particularly interested in Ringo C, which is expressed in numerous human tissues that undergo endoreplication (S phase in the absence of cell division) and are exploring its role in this process.

The Anaphase-Promoting Complex (APC)

We are very interested in ubiquitin-mediated proteolysis involving the Anaphase-Promoting Complex (APC), which promotes the degradation of mitotic cyclins and a key inihibitor of anaphase onset. We have found that the APC activators Cdc20 and Cdh1 directly bind substrates via substrate degradation motifs termed the Destruction Box and the KEN Box. Binding of a Destruction Box targets the Cdh1-substrate complex to the APC. The Spindle Assembly Checkpoint prevents APC-mediated degradation during mitosis when even a single chromosome is not properly attached to the mitotic spindle. We found that a key mediator of this checkpoint, Mad3, binds to Cdc20 via conserved KEN Boxes and a Destruction Box. Unlike APC substrates, Mad3 is not ubiquitinated. Rather, this pseudosubstrate binding of Mad3 to Cdc20 prevents substrates from binding to Cdc20. We believe that this competitive inhibition provides the basis for the spindle checkpoint and we have been able to unify our model with previous models attempting to explain how the checkpoint works.