David L. Brautigan, Ph.D.
Professor of Microbiology, Immunology, and Cancer Biology
Research: Protein Phosphorylation in Cell Signaling
OVERVIEW: Our goal is to discover molecular mechanisms of intracellular signaling that regulate cell proliferation, and survival/apoptosis, 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, with techniques of biochemistry and cell and molecular biology. It is typical for students and fellows to learn and use the full array of modern techniques 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.
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 propose that PP6 is a trimeric enzyme, composed of ARS, SAPS and a catalytic subunit. The 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 at limiting density and the numbers of colonies arising after 30 days counted and scored relative to controls, plotted on a logarithmic scale. Loss of PP6c or SAPS1 was equivalent to loss of DNA-PK itself.
Protein Phosphatase-6 is especially abundant in the gastrointestinal tract. In confluent monolayers of human Caco-2 intestinal epithelial cells that are a well-accepted in vitro model of the human intestine we visualized PP6 localized at cell-cell junctions by immunofluorescent confocal microscopy, in particular at adherens junctions with E-cadherin, not at tight junctions with ZO-1 occludin. PP6C and E-cadherin were co-immunoprecipitated to show interaction of the endogenous proteins in epithelial cell membranes. Using inducible lentivirus constructs to switch on shRNA and knockdown PP6C there was a dramatic dissolution of E-cadherin localization from cell-cell junctions (see red staining in panel (B) Figure below). This effect was due to internalization of the E-cadherin, in response to phosphorylation of E-cadherin at a CK1 site at S846 in the cytoplasmic tail of the protein. Treating cells with IC-261, a CK1 inhibitor blocked phosphorylation of this site in E-cadherin and prevented internalization.
To analyze the images we used line scanning confocal microscopy across the cell-cell boundaries (panel C. below), fitted the pixel intensities to Gaussian curves and then determined the Full Width at Half Maximum (FWHW). This analysis allows us to demonstrate statistically significant changes the spread of the E-cadherin from the junctions - due to RNAi knockdown of PP6c and reversal due to IC-261. We propose that PP6 regulates the surface expression and internalization of E-cadherin in epithelial cells.
RhoGDI2 in Bladder Cancer and Metastasis
Bladder cancer cells have been studied in collaboration with Dan Theodorescu, 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. 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. Current work is examining how the regulatory protein RhoGDI2 functions. RhoGDI2 associates with and blocks the actions of a select subset of GTPases, and we are testing the hypothesis that these provide signaling to support metastatic disease.
|Purifying phosphatases using column chromatography guided by enzyme assays in the Center coldroom.|
Most recent update February, 2014.