A focus of interest in lipid-based signaling in the Topham-Stafforini Lab is the examination of the fate of the intracellular messenger diacylglycerol (DAG). DAG accumulates transiently in cells exposed to growth factors or other stimuli. Elevated DAG mediates diverse cellular responses such as growth and differentiation by virtue of binding to and activating protein kinase C. This process can be activated inappropriately in cancer cells, as these cells often have high levels of diacylglycerol.
The Topham-Stafforini Lab has provided key insights into the regulation of this process by identifying and cloning several genes that encode diacylglycerol kinases, enzymes that shut off the DAG activation signal. Moreover, they have shown that one of the DGK isoforms has a novel mechanism for shuttling in and out of the nucleus and that while in the nucleus it suppresses growth signals. The discovery of this large family of lipid kinases has opened many new studies of signaling mechanisms and their cellular consequences.
Within the lab, Matthew K. Topham, MD, Assistant Professor, Department of Internal Medicine, directs research focused on the diacylglycerol kinases. Topham has been supported as a fellow of the Howard Hughes Medical Institute and has been an investigator at Huntsman Cancer Institute and a member of the faculty of the University of Utah Department of Internal Medicine since 1998.
Diacylglycerol kinases (DGKs) are responsible for "turning off" the function of diacylglycerol (DAG), an important molecule that is often abnormally active in cancer cells. They also produce phosphatidic acid (PA), a molecule that can cause abnormal cell division, leading to cancer. Thus, this large family of lipid kinases occupies an important biological role and their activity must be tightly regulated. This biologic mechanism is relevant to many types of cancer, but Topham has a particular interest in its relationship to lung and breast cancer.
A recent report from Topham's group focuses on the role of a particular DGK, DGKzeta, in the regulation of factors that promote activity of the oncogene product Ras*. Ras activity must be precisely regulated or abnormal cellular proliferation can occur. An estimated 30% of human tumors have an activating mutation of the Ras gene. Guanine nucleotide exchange factors (GEFs) activate Ras by facilitating GTP binding. Such an exchange factor, RasGRP, was recently identified as a potential leukemia disease gene and overexpression of this protein in cultured cells induces a transformed phenotype. Combined, these observations indicate that abnormally high RasGRP activity can lead to malignant transformation.
RasGRP has a diacylglycerol (DAG)-binding domain and its exchange factor activity depends on local availability of the signaling molecule DAG. DAG kinases (DGKs) remove DAG from the cell by converting DAG to PA. Because they can attenuate local accumulation of DAG, Topham hypothesized that DGKs might serve as an "off" mechanism for RasGRP and, consequently, reduce activation of Ras. By attenuating the DAG pool necessary to maintain RasGRP activity, DGKs may have a pivotal role in modulating Ras signaling in some contexts.
Topham found that DGKzeta, but not other DGKs, completely eliminated Ras activation induced by RasGRP, and that diacylglycerol kinase activity was required for this mechanism. DGKzeta also co-immunoprecipitated and co-localized with RasGRP, indicating that these proteins associated in a signaling complex. Co-immunoprecipitation of DGKzeta and RasGRP was enhanced in the presence of phorbol esters, which are DAG analogues that cannot be metabolized by DGKs, suggesting that DAG signaling can induce their interaction. These results suggested a model in which the activity of RasGRP, and consequently Ras, is exquisitely regulated by the coordinated activity of enzymes that generate DAG, such as PLCg1, and DGKzeta, which terminates the DAG signal.
Topham's work demonstrated that DGK regulation appears to be selective and spatially discrete: only one of six DGK isotypes, DGKzeta, inhibited RasGRP. Based on his data, it appears likely that each DGK isoform has the specific function of regulating one or a few lipid-activated proteins by locally metabolizing DAG or by generating PA. This regulation may represent a general mechanism in which a DGK associates with a protein activated by DAG and regulates its activity through its DGK enzymatic function. Potentially, it may be possible to affect signaling events important for tumorigenesis by altering DGK activity.
*Topham MK, Prescott, SM: Diacylglycerol kinase zeta regulates Ras activation by a novel mechanism. J Cell Biol. 152: 1135-1143, 2001.