Dr. Jeffrey Singer
One of the major protein degradation pathways found in eukaryotic cells is the ubiquitin degradation system. Ubiquitin mediated protein degradation utilizes a sequential cascade of enzymes, called E1 through E3, that result in the addition of ubiquitin to the substrate that is targeted for degradation. E3s, or ubiquitin ligases, are proteins that are responsible for recognition of the substrates targeted for degradation. One important class of E3s are the cullins that function as parts of multi-subunit complexes that are assembled in a modular fashion. Cullins have been shown to be involved in a large variety of biological processes, including cell cycle control, removal of N-glycosylation containing proteins, transcriptional control, hormonal regulation, differentiation, development, and prevention of neurological disorders. Our laboratory is interested in understanding how E3 ligases, the substrate recognition activity, function in mammalian cells. Our initial work has identified an E3 ligase that contains a cullin called Cul3 as a regulator of cyclin E. A targeted knockout of the Cul3 gene in mice resulted in early embryonic lethality with some cells exhibiting elevated levels of cyclin E compared to controls. Since not all cells exhibited this phenotype and elevated levels of cyclin E have not been shown to cause embryonic lethality, we hypothesized that Cul3 was involved in other important cellular processes. In order to determine what these processes are, we constructed a conditional knock out of the Cul3 gene, a floxed allele, which allows us to reduce Cul3 levels in a selective manner. We are taking four different experimental approaches to address the function of Cul3:
We are using two-dimensional gel electrophoresis to identify all the proteins that are bound to Cul3. Immunoprecipitation of Cul3 complexes from radioactive extracts has revealed about 20 spots when analyzed using gels. We are also using gel exclusion chromatography to isolate in vivo complexes from cells. Our initial data shows that Cul3 exists in several different sized complexes. We have recently developed a TAP tagged Cul3 construct and have identified a large number of novel Cul3 binding proteins as well.
BTB domain containing proteins
We have isolated a large number of BTB containing proteins that bind Cul3 and propose that they function as substrate selectors for the Cul3 E3 ligase. We are presently studying the function and expression patterns of these adaptors.
Using a floxed Cul3 allele, we are making extracts from large amounts of cells that lack Cul3. We are using 2-D gels to analyze the differences in protein levels between wild type cells and those that lack Cul3. We believe that this approach will allow us to get a complete picture of all of the Cul3 substrates. We are also using this allele to determine when and where Cul3 is required for embryonic development and adult proliferative responses.
Cul3 and cancer
Cyclins E and D have been show to be elevated in primary breast tumors and breast tumor cell lines. We have shown that tumors with elevated levels of cyclin E have reduced levels of Cul3. We have also shown that mice lacking Cul3 arrest during embryonic development with excess cyclin E. We therefore propose that Cul3 acts as a tumor suppressor by controlling levels of cyclins that are limiting for cell proliferation. We have developed a mouse model to look at the loss of Cul3 in the formation of breast tumors.
- Wimuttisuk, W. and Singer, J.D. (2007). The Cullin3 Ubiquitin Ligase Functions As A Nedd8-Bound Heterodimer. Mol. Biol. Cell. 18: 899-909.
- McEvoy, J.D., Kossatz, U., Malek, N., and Singer, J.D. (2007). Constitutive turnover of cyclin E by Cul3 maintains quiescence. Mol. Cell Biol. 27(10): 3651-3666.
- Watai, Y., Kobayashi, A., Nagase, H., Mizukami, M., McEvoy, J. D., Singer, J. D., Itoh, K. and Yamamoto, M. (2007). Subcellulええvれえvれう゛ぇあrfo complexirect complex formation of endogenous Keap1-Nrf2-Cul3 complex-Tublin antibodies. derived from Cul3 conditar Localization and Cytoplasmic Complex Status of Endogenous Keap1. Genes to Cells, 12(10): 1163-1178. (Featured on cover).
- Cummings, C., Bentley, C.A., Perdue, S.A., Baas, P., and Singer, J.D. (2009). The Cul3/KLHDC5 E3 Ligase Regulates P60/Katanin And Is Required For Normal Mitosis In Mammalian Cells. J. Biol. Chem., 284(17): 11663-75.
- Plafker, K.S., Singer, J.D., and Plafker, S.M. (2009) The ubiquitin conjugating enzyme, UbcM2 , engages in novel interactions with components of cullin-3 based E3 ligases. Biochem., 48(15): 3527-37.
- Claiborn, K.C., Sachdeva, M.M., Groff, D.N., Singer, J.D. and Stoffers, D.A. (2010). Pdx1 regulation of pancreatic beta cell survival and function is modulated by the ubiquitin ligase adaptor Pcif1. Journal of Clinical Investigation, 120 (10): 3713-3721.
- Kossatz, U., Breuhahn, K., Wolf, B., Steinemann, D., Singer, S., Brass, F., Kubicka, S., Schlegelberger, B., Schirmacher, P., Manns, M.P., Singer, J.D., and Malek, N.P. (2010). The cyclin E regulator cullin 3 prevents mouse hepatic progenitor cells from becoming tumor initiating cells. Journal of Clinical Investigation. 120 (11): 3820-3833.
- Marshall, J., Blair, L. A. C., and Singer, J.D. (2011) BTB-Kelch Proteins and Ubiquitination of Kainate Receptors. Pp. 115-125. In Kainate Receptors (Rodriquez-Moreno, A., and Sihra, T. eds.), Landes Scientific.
Picture of a Cul3 dimer