Checkpoint Control in Mitosis
Research in Dr. FangDs laboratory focuses on the role of proteolysis in control of biological transitions. Currently, we are investigating the regulation of cell cycle progression by the ubiquitin-dependent proteolysis pathway and the mechanism of the spindle assembly checkpoint control. We are also interested in studying the role of proteolysis in cell differentiation and in the pathogenesis of certain diseases. The long-term goal of our work is to understand the biochemistry of these proteolysis pathways and the biology of the processes they control. To achieve this, we use multidisciplinary experimental approaches, including biochemical, cell biological, and genetic analyses in Xenopus cell-free extracts and mammalian tissue culture cells.
Ubiquitin-dependent proteolysis requires three enzymes, a ubiquitin-activating enzyme, a conjugating enzyme, and a ligase, which act sequentially to transfer ubiquitin to target proteins. Poly-ubiquitinated proteins are subsequently degraded by proteasomes. Intracellular proteolysis not only is utilized for the degradation of mis-folded proteins, but also can control discrete biological transitions in the cell cycle, signal transduction and development. Proteolysis is an irreversible process that can act as a binary, all-or-none switch to control biological transitions.
We are currently studying the control of cell cycle transitions by proteolysis. In mitosis, a ubiquitin-dependent degradation pathway controls two critical transitions: the metaphase to anaphase transition (through degradation of the anaphase inhibitor) and exit from mitosis (through degradation of mitotic cyclins). The key component of this mitotic degradation machinery is a ubiquitin ligase, the anaphase-promoting complex (APC). APC controls multiple events in mitosis and in G1 and is a master regulator of the cell cycle. Thus, control of the APC activity towards different substrates in a specific temporal manner is crucial for the ordered events of mitosis. We have shown that the activity of APC is regulated by a combination of phosphorylation of APC subunits and binding of regulatory co-factors, CDC20 and CDH1. We are investigating what controls the phosphorylation of APC and how the activities of CDC20 and CDH1 are regulated through the cell cycle.
The progression of mitosis is monitored by a surveillance mechanism, the spindle assembly checkpoint. This checkpoint mechanism monitors the attachment of kinetochores to the mitotic spindle; the presence of a single unattached kinetochore activates the checkpoint mechanism, which transduces a signal to inhibit anaphase initiation. The spindle assembly checkpoint mechanism ensures the fidelity of sister chromatid separation. Aneuploidy could arise from inactivation of this checkpoint pathway, and indeed the spindle assembly checkpoint pathway is defective in certain types of cancers, such as T cell leukemia and colorectal cancers. Thus, investigating the biochemistry of the checkpoint pathway could lead to more effective strategies for cancer prevention, diagnosis and treatment. Since activation of APC is required for the metaphase to anaphase transition, APC is a key target for checkpoint intervention. We have shown that the checkpoint protein MAD2 directly binds to CDC20 and together they form a ternary complex with APC. MAD2 inhibits APC in the ternary complex and prevent pre-mature separation of sister chromatids prior to anaphase. We are currently investigating how the checkpoint signal is generated at unattached kinetochores and how the signal is transduced, leading to inhibition of APC.
Proteolysis by the APC pathway has also been implicated in cell differentiation. CDH1 and APC are expressed at high levels in post-mitotic, terminally differentiated neurons and APC activity in these cells is maintained by the associated CDH1 protein. Similarly, APC is active in myotubes differentiated from C2C12 myoblasts in vitro. We will investigate the function of APC through genetic analyses in tissue culture cells and in mouse as well as through identification of APC substrates in differentiated neurons and myotubes.
Proteolysis controls critical cellular functions, and de-regulation of proteolysis pathways can lead to pathogenesis of certain diseases. The APC pathway is the best studied ubiquitination pathway and has provided much of our current knowledge on how proteolysis controls biological transitions. A better understanding of the regulation of APC and checkpoint control mechanism is central not only to our understanding of the basic cell cycle machinery, but also to our knowledge of the biological control of proliferation and differentiation in general.