A central theme of the research in this laboratory focuses on the identification and regulation of genes important for progression through the eukaryotic cell cycle. We are interested in how human cells decide when to synthesize DNA. Using novel expression strategies, we have identified a protein kinase, Cdk2, which appears to be the central cell cycle kinase that binds cyclin A and cyclin E and regulates entry into S-phase. We have pursued the regulation of Cdk2 and together with Dr. Wade Harper identified a novel class of cell cycle regulators of Cdks called CKIs, Cdk inhibitors. The first of which was p21CIP. There are now three members of the p21 family, p21, p27 and p57 discovered in our lab and others. Work from our lab and others have determined that p21 is transcriptionally regulated by p53, is induced in response to DNA damage and controls the G1 checkpoint in response to DNA damage. p57 is genomically imprinted, being expressed only from the maternally inherited chromosome. p57 plays a role in controlling development of certain tissues. Recently, we and others have found mutations in the human p57 gene in a familial cancer syndrome BWS (Beckwith-Weidemann's Syndrome).

We have explored the role of these proteins in the control of cell proliferation during mouse development and have found that they have both unique roles and redundant roles. p21 deficient mice are developmentally normal. p27 deficient mice display overgrowth and multi-organ hyperplasia. p57 has a role in the control of cell proliferation in limb growth, palate formation and in the lens. p21 and p57 play a redundant role in lung and muscle differentiation in vivo. p27 and p57 play redundant roles in lens development.

Novel Cyclins

Expression cloning has allowed the identification of two novel human cyclins, cyclin F, an S/G2 cyclin, and cyclin K. The cyclin F protein accumulates in interphase and is destroyed at mitosis at a point distinct from cyclin B. Cyclin F shows regulated subcellular localization and overexpression of cyclin F resulted in a significant increase in the G2 population implicating cyclin F in the regulation of cell cycle transitions. We do not yet know its function in the cell cycle. Cyclin K is a member of the transcription cyclin family and is associated with a kinase activity capable of phosphorylating the tail of RNA polymerase and Cdks on an activating threonine in the T-loop.

Mammalian DNA Damage Checkpoints

We have previously shown that the Cdk inhibitor p21 is induced in response to DNA damage and is required for cell cycle arrest in G1. Apparently there is also a role for tyrosine phosphorylation of Cdk4 that is involved in G1 arrest as well that is not currently well understood.

We and others have identified the mammalian homologs of several yeast checkpoint genes. MEC1 is a homolog of the human ATM gene involved in human cancer. We identified the human Chk1 kinase and found that it is modified and presumably activated in response to DNA damage. Together with the Piwnica-Worms lab we showed that Chk1 phosphorylates the human Cdc25C phosphatase on an inhibitory serine leading to its inactivation. Cdc25C is an activator of the Cdc2 kinase needed for mitotic entry and this connection establishes a mechanism for G2 arrest in mammals in response to DNA damage. We have also shown that Chk1 can phosphorylate Cdc25A and Cdc25B and may therefore be involved in regulating other cell cycle transitions in addition to G2.

We are interested in identifying other homologs of known cell cycle proteins and working out the biochemistry of their regulation in response to DNA damage.