David L. Brautigan, Ph.D.
Professor of Microbiology, Immunology, and Cancer Biology
Research: Protein Phosphorylation and Ser/Thr Phosphatases in Cell Signaling
OVERVIEW: Our goal is to discover how intracellular signaling pathways regulate cell proliferation, and survival/apoptosis in cancer. 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 cells and bacteria to combine functional genomics, biochemistry and cell biology. It is typical for students and fellows to learn and use the full array of molecular and cellular techniques while studying these signaling networks. (e.g. PCR, cloning, mutagenesis, protein expression and purification, tissue culture, transfections, enzyme assays, immunoprecipitations, immunoblotting, microscopy, etc).
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, see Figure). On the other hand, protein dephosphorylation is catalyzed by different families of protein phosphatases: PTPs (yellow), PPPs (orange), PPMs (green) and HADPs (purple) that have different structures, different active sites and different catalytic mechanisms (Brautigan, 2012 FEBSJ).
The PPP phosphatases have bimetallic actives sites with Fe and Zn (or Mn) and are related in sequence, structure and mechanism to the purple acid phosphatases. 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 Phosphatase-6 in Cell Cycle, DNA damage
responses and Epithelial Differentiation
Protein Phosphatase 6 (PP6) is a distinct member of the protein Ser/Thr phosphatase family that is the mammalian homologue of yeast 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).
Structural models for SAPS domain in PP6R3. The sequence of PP6R3 residues 1-513 was used to produce alternate models based on known helical-repeat proteins (left to right): A- PP2A A subunit, B- beta-catenin, C- p115 golgin. Predicted alpha helices are shown in red (convex) and blue (concave), with intervening loops as strands. Side chains of E63, E64, D113, E204, E205, E259 and E262 are shown as space-filling models in yellow.
Proteomics of immunoprecipitated SAPS complexes using mass spectrometry here at UVA revealed 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 now propose that PP6 is a trimeric enzyme, composed of ARS, SAPS and a catalytic subunit. We have analyzed the structure of SAPS subunits using modeling, to predict a helical repeat arrangement, and find charged residues that are needed for PP6 association (BMC Biochem. 2009). A project is underway to crystallize and determine the 3D structure of the SAPS domain.
DNA-PK, is a Ser/Thr kinase activated following damage to DNA and an initiator of DNA repair by the non-homologous enjoining (NHEJ) pathway. We found that in glioblastomas (brain tumor cells) PP6 and one of its SAPS subunits we call PP6R1 were recruited into the nucleus and into complexes with DNA (PLoS One; 2009) PK. 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 (gamma rays), and for the repair of double strand breaks in DNA, and for the survival of the cells . Thus, inhibiting this action of PP6 makes cells more sensitive to radiation, and may provide new therapies to enhance radiation therapy for otherwise incurable brain tumors. These results led us to generate SAPS1 knockout mice that we are now studying.
Recently we observed that PP6C in human intestinal epithelial cells so visualized by confocal microscopy is localized at cell-cell junctions, in particular at adherns junctions with E-cadherin, not at tight junctions with ZO-1. 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. This effect was due to internalization of the E-cadherin, in response to phosphorylation of a CK1 site at S846 in the cytoplasmic tail of the protein. We are pursuing the signaling pathways linking PP6C and E-cadherin proteins.
Breast Cancer and Cyclin D1 Phosphatase
Clinical studies of human breast cancers reveal that the protein cyclin D1 (D1) predicts poor tumor response to tamoxifen, the widely used anti-estrogen chemotherapy. Half of all breast tumors have elevated D1 and those high levels of D1 confer resistance to tamoxifen. Ablation of D1 in animal models by gene knockout or gene silencing arrests cancer cell growth and prevents formation of breast tumors, but in 10 years these discoveries have not translated into clinical treatments. There are still not effective ways to delete or silence specific genes in patients who have cancer. New approaches are needed to eradicate breast cancers by reducing levels of D1.
Other studies of D1 in cancer cells reveal it is constantly consumed by a disposal pathway that is triggered by phosphorylation of D1, a reaction catalyzed by GSK3 and MAPK kinases and reversed by a Ser/Thr phosphatase. Simply put, more phosphorylation of D1, less D1 protein survives. We tested cell-permeable phosphatase inhibitors on human breast cancer cells and found calyculin A increases D1 phosphorylation and causes the cells to rapidly degrade all their D1 and stop growing (Toxins, 2011).
This offers an entirely new approach to treat breast cancer, including, especially, tamoxifen-resistant tumors, by developing a class of drugs that will act as D1 phosphatase enzyme inhibitors. We are working to isolate and determine the subunit composition of the D1 phosphatase and screening libraries of chemical compounds to find novel inhibitors of this phosphatase that could mimic (and replace) calyculin A. Our discovery-based research represents the critical early steps toward the goal of new medicines for breast cancer.
Our work is supported by grants from the United States Public Health Service (USPHS) - National Institute of General Medical Sciences, as well as the UVA Cancer Center, made possible in part by philanthropic donations.
|Purifying phosphatases using column chromatography guided by enzyme assays in the Center coldroom.|
Most recent update February, 2013.