Research Interest

Research Interest

David L. Brautigan, Ph.D.
F. Palmer Weber Medical Research Professor

Department of Microbiology, Immunology, and Cancer Biology
Center Director

Research: Protein Phosphorylation in Cell Signaling

OVERVIEW: The goal of our research is to discover molecular mechanisms of intracellular signaling that regulate cell survival and proliferation, especially in cancer cells. Knowledge of these signaling events, and the enzymes involved, provides the basis for understanding normal physiology as well as the diagnosis, therapeutic treatment and even prevention of human diseases.  We primarily use cultured human cell lines to pursue functional genomics. It is typical for students and fellows to learn and use the full array of modern techniques of biochemistry and cell and molecular biology while studying these signaling networks. (e.g. PCR, cloning, mutagenesis, tissue culture, RNAi, transfections, protein expression and purification, enzyme assays, immunoprecipitations, immunoblotting, fluorescent microscopy).

Our focus is on protein phosphorylation that controls essentially every process in human cells. Phosphorylation on Ser/Thr residues accounts for >95% of cellular phosphoproteins, the other 5% is Tyr phosphorylation. There are about 500 protein kinases in the human genome, all in one superfamily with conserved 3D enzyme structure and common mechanism of action (blue box, see Figure below). On the other hand, protein dephosphorylation is catalyzed by multiple families of protein phosphatases, each quite different from one another: PTPs (yellow), PPPs (magenta), PPMs (green) and HADPs (purple) that have different structures, different active sites and different catalytic mechanisms (Brautigan, 2013 FEBS Journal, PMID 22519956). The PPP phosphatases have bimetallic actives sites with Fe and Zn (or Mn) and are related in sequence, structure and mechanism. Genomics has shown that the PPP enzymes are extraordinarily conserved in all eukaryotes (e.g. mammals, Xenopus, Drosophila, C. elegans, S. pombe, S. cerevisiae). Humans and yeast have about the same total number of PPP genes, in separate functional classes (i.e. PP1, PP2A, PP4, PP6). These classes of PPP are all sensitive to inhibition by nanomolar concentrations of toxins such as calyculin A, okadaic acid, microcystin and cantharidin produced by marine dinoflagellates, or blue-green algae, or insects. We use these toxins as experimental tools. Individual human PPP proteins can substitute in place of their yeast homologues, but not PPP of other functional classes, showing that individual PPP are functionally equivalent across evolution, but each class has distinctive biological actions. The conservation across species allows us to use the results from genetic experiments in various model organisms to guide our study the human versions of PPPs.

protein phosphorylation

Functions of Protein Phosphatase-6 and SAPS

Over the past decade we have focused on Protein Phosphatase 6 (PP6), a distinct member of the protein Ser/Thr phosphatase family that is the mammalian homologue of yeast S. cerevisiae Sit4. The functions of Sit4/PP6 are conserved, because human PP6 rescues yeast sit4- mutations, whereas other PPP do not. In yeast Sit4 has associated subunits called SAPS and we were the first to clone and characterize three humans SAPS as specific subunits of PP6 phosphatase (J. Biol. Chem. 2006). We found that expressing GFP-PP6 compared to GFP-PP2A has effects on G1 to S phase progression in human prostate cancer cells, influencing the levels of cyclin D1 and phosphorylation of Rb (Cell Cycle, 2007). 

Other evidence points to PP6 in cytokine signaling and pathways leading to activation of NF-kB. PP6 SAPS subunits mediate association with IkBe and alter the degradation of this regulator in response to TNF stimulation (J. Biol. Chem. 2006).  PP6 regulates activation of the TAK1 kinase, through interaction with the protein TAB2 (J. Biol. Chem. 2010).

We carried out proteomics of immunoprecipitated SAPS complexes using mass spectrometry and discovered that these subunits bind a family of Ankyrin Repeat Subunits (we named ARS) that are functionally equivalent to the SAPS themselves in siRNA knockdown assays (Biochemistry, 2008). Thus, we proposed that PP6 is a trimeric enzyme, composed of ARS, SAPS and a catalytic subunit. These results have been independently confirmed by other laboratories. We have analyzed the structure of SAPS subunits and used molecular modeling to predict a helical repeat arrangement, and mutated charged residues that are needed for PP6 association (BMC Biochem. 2009).  Another protein associated with SAPS1 was DNA-PK, a Ser/Thr kinase activated following damage to DNA and an initiator of DNA repair by the non-homologous end joining (NHEJ) pathway.

In a long-standing collaboration with Dr. James Larner, Chairman of the Department of Radiation Oncology, we found that in glioblastomas (brain tumor cell lines) PP6 and one of its SAPS subunits (a.k.a. PP6R1) were recruited into the nucleus and into complexes with DNA-PK (PLoS One; 2009). We used deletions and co-immunoprecipitation to show that SAPS1 depends on two separate regions to form complexes with DNA-PK (J. Biol. Chem. 2012).

PP6 and PP6R1 both were required for the activation of DNA-PK following ionizing radiation (see Figure below), and for the repair of double strand breaks in DNA, and for the survival of the cells. We used sequence-specific RNAi to deplete the cells of different proteins, and confirmed the knockdown by immunoblotting.  Thus, reduction in the amount of PP6, or SAPS1, makes cells more sensitive to radiation, and may provide an opportunity for new therapies to enhance radiation therapy for otherwise incurable brain tumors. These results led us to generate SAPS1 conditional knockout mice that we are now studying.



Clonal survival of human glioblastoma multiforme (GBM) cells exposed to increasing doses of ionizing radiation. GBM cells were transfected with siRNA to knockdown expression of PP6 catalytic subunit (solid circles) or SAPS1 subunit (blue squares ) or control siRNA (open circles) and compared to an isogenic cell line lacking DNA-PK (red diamonds). Following radiation cells were plated and the numbers of surviving colonies scored as % of control, plotted on a logarithmic scale. Loss of PP6c or SAPS1 was equivalent to loss of DNA-PK itself.


Androgen Receptor (AR) ablation in prostate cancer.

Prostate cancer is a major health challenge that impacts everyone, by afflicting about one in six American men. Hormone deprivation is a primary approach to therapy, depriving the androgen receptor (AR) of its activating ligand to limit tumor growth.  However, tumor cells still survive because of mutations in the AR, or by AR-dependent signaling in the absence of ligand. Jim Larner and I are interested in promoting the degradation of the AR protein in cancer cells, which would potentially arrest tumor growth. UVA investigator George Amorino, who died from cancer at a young age, found that the metabolite 2-methoxyestradiol (2-ME) a compound tested as an anti-tumor agent itself, caused a synergistic enhancement of radiation-induced tumor regression in xenograft mouse models (Cancer Res. 2007; see Figure). The 2-ME alone (solid squares) had no effect on tumor growth compared to untreated controls (open squares), and radiation alone (open circles) gave some delayed growth inhibition, compared to the combination of 2-ME plus radiation that stopped the tumors from growing (solid circles).



Effect of combined radiation and 2-ME treatment on tumor growth. Nude mice with subcutaneous PC3 tumors (hind leg) were treated +/- 2 Gy for 5 consecutive days; 2-ME (75 mg/kg, closed symbols) was administered orally 4 h prior to each radiation dose.  Tumors were measured by luciferase light emission in terms of photon flux. Error bars represent  ±SEM for 5 mice per treatment group.


We studied this effect of 2-ME and found by FACS that it involved cell cycle arrest of the cells at G2/M. There was a loss of the AR from the cells and we went on to find 2-ME induces this time-dependent loss of AR from human prostate cancer cells via ubiquitin-mediated proteolysis, which is inhibited by adding the drug MG-132 (see Figure). (Oncogene, 2014).

Time course of androgen receptor (AR) degradation induced by addition of 2-methoxyestradiol (2-ME) and inhibited by MG-132 to show dependence on proteosomal activity. Western blot of AR, and GAPDH as loading control.



Using RNAi to knockdown different proteins we showed that degradation of AR dependent on hsp70 and its partner CHIP, an E3 ligase that conjugates ubiquitin to AR. More recent data indicates this involves activation of the process in a phosphorylation-dependent mechanism. We are now pursuing which target proteins, such as CHIP, AR, hsp70, etc. are involved and what signaling pathways and kinases are triggered by 2-ME.

RalGTPase and RhoGDI2 in Bladder Cancer and Metastasis

In collaboration with Dan Theodorescu, former UVA Professor who is now Director of the University of Colorado Cancer Center we found phosphorylation by PKC activates the RalB GTPase (Cancer Research, 2010).  The Rho family GTPases including RalA/B control a diverse range of cellular processes, and their deregulation has been implicated in human cancer.  We also discovered small molecule inhibitors of Ral that are effective in ELISA assays, cell-based assays and in vivo tumor xenografts (Nature, 2014). These compounds have been licensed to a biotechnology company for further clinical development as oncology drugs.

RhoGDI2 is a guanine nucleotide dissociation inhibitor (GDI) that binds and sequesters different GTPases in the cytosol, restricting their activation. RhoGDI2 acts as a metastasis suppressor in bladder cancer, and reduction of RhoGDI2 expression correlates with tumor grade and stage and is a predictor for the development of bladder cancer metastasis and poor prognosis for patients (Griner and Theodorescu, 2012).  While RhoGDI2 is thought to bind specifically to the GTPase Rac1 to negatively regulate Rac1 activation, no study has been able to show changes in Rac1 activation levels in bladder cancer cells upon introduction of exogenous RhoGDI2, indicating that RhoGDI2 may be exerting its metastasis suppressor effects through an as yet unidentified binding partner. Using proteomics and quantitative immunoprecipitations (shown here) we showed that RhoC in addition to Rac1 was a major endogenous partner of RhoGDI2.

RhoC activation was regulated by expression or knockdown of RhoGDI2. RhoC knockdown reduced bladder cancer cell proliferation, as well as invasion through Matrigel and colony formation in soft agar, as surrogate assays for metastasis. These results were extended to in vivo experiments, where RhoC knockdown reduced lung metastases following tail vein injection of tumor cells. AffyMetrix gene arrays revealed sets of genes that were regulated by both RhoC and RhoGDI2. Our results (in Molec.Cancer Res., 2014) provide new support for the key role of RhoC in promoting metastasis of tumors.


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Most recent update February, 2015.