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David L. Brautigan, Ph.D.
Professor of Microbiology and Internal Medicine; Center Director
Protein Ser/Thr Phosphatases in Cell Signaling
Our goal is to discover how intracellular signaling pathways control the cell cycle and mitosis, and regulate cell survival/apoptosis and cytokine production in response to stress signals and infections. This research is relevant to understanding normal physiology as well as the pathology encountered in human diseases such as cancer, diabetes and autoimmunity. We primarily use tissue culture cells and combine functional genomics, biochemistry and cell biology. It is typical for students to learn 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, and especially the enzymes called protein Ser/Thr phosphatases (the PPP and MPP Families). Phosphorylation on Ser/Thr accounts for >95% of cellular phosphorylation, the other being Tyr phosphorylation by receptors and oncogenes (mostly in metazoans). There are about 500 protein kinase in the human genome, all in one super family with conserved 3D structure. On the other hand protein dephosphorylation is done by more than three protein families of phosphatases PTPs, PPPs and MPPs that have different structures, active sites and different catalytic mechanisms. (see Figure)
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 produced by marine dinoflagellates, or blue-green algae, or insects. 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 a distinctive biological role. This allows us to use the results from genetic experiments in various model organisms to guide our study the human versions of PPPs.
Cell Cycle and Mitosis
The most ancient of the PP1 phosphatase regulators is inhibitor-2 (I-2), which is expressed in all eukaryotes (called GLC8 in yeast) and such evolutionary conservation typically implies important biological function. For the 30 years since its discovery it has been considered an inhibitor of protein phosphatase PP1, as its name indicates, however, we have discovered new and unexpected properties that demonstrate I-2 is a multifunctional protein important for mitosis. Immunofluorescent microscopy in this lab showed I-2 localizes at centrosomes. I-2 inhibits PP1 that is complexed to the kinase Nek2 at centrosomes to affect centrosome separation in the process of forming spindles to separate chromosomes at mitosis (J. Biol. Chem. 2002). Nek2 and PP1 form a kinase-phosphatase molecular switch by inactivating one another while self-activating by phosphorylation and dephosphorylation.
Craig Leach completed his Ph.D. in the Cell and Molecular Biology Program in 2003 and as part of his thesis he made a phospho-site specific antibody against P-Thr72 in I-2, a residue conserved in a PxTP sequence motif in all various species, and found that this site is selectively phosphorylated during mitosis (J. Biol. Chem., 2003). Craig used recombinant I-2 protein he expressed in bacteria, plus lysates made from interphase or mitotic HeLa cells, in a test-tube assay to detect I-2 kinases during mitosis. Postdoc Mingguang Li used this assay with cells synchronized in the cell cycle to show mitotic cyclin B1:CDC2 is the primary I-2 kinase in cells and used the phospho-T72-I-2 antibody to selectively stain mitotic cells (Cell Signaling, 2006).
This phosphosite PxTP sequence motif in I-2 is similar to other cyclin B1:CDC2 substrates and these are recognized by a conserved prolyl isomerase called Pin1 (ESS1 in yeast) that alters the conformation of the backbone PThr-Pro bond to affect protein function. Mingguang found that Pin1 did not react with the PxTP site in I-2, however I-2 did bind to Pin1 to act as a regulatory subunit, changing Pin1 specificity for different mitotic phosphoproteins (Biochemistry, 2007). This leads us to propose a model for cell cycle regulation of Pin1 by I-2, controlled by phosphorylation of both proteins. This model needs to be tested and the actions of Pin1 with I-2 need to be further defined. Thus, I-2 seems to be a multitasking protein, not simply a PP1 inhibitor. Its conservation during evolution probably is a consequence of having multiple actions in cells and being critical to mitosis. This idea is supported by work done by graduate student Weiping Wang, who discovered I-2 is required for chromosome segregation by RNAi knockdown in human cells and in hypomorphic Drosophila embryos (Molec. Bio. Cell, 2008; Genetics, 2008).
Drosophila embryo deficient for I-2 loses mitotic sydmony. |
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Migration and Cancer
Protein phosphatase-1 (PP1) has multiple roles in control of the cytoskeleton, cell migration and cancer cell survival. Using isoform-specific antibodies we prepared we discovered that PP1alpha is targeted to a subset of focal adhesions by binding to the protein tensin, which binds to integrins and anchors the actomyosin stress fibers in cells. We co-precipitated PP1alpha and tensin (Figure from J. Biol. Chem. 2006). As cells spread or migrate they form adhesions along the leading edge that can be stained with vincullin (red) and these adhesions convert to larger, force generating adhesions that contain tensin and PP1alpha (green; yellow in merged image). Metastatic breast cancer cell lines are deficient in tensin providing a system to study tensin function. Postdoc Emily Hall is expressing mutated tensins to study the effects on cancer cell migration and binding to PP1 appears necessary for tensin to regulate polarity and motility.
Human Breast Cancer MCF7 Cells |
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Cytokines and Inflammation
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 is genetically linked to cell cycle control. We have found that PP6 has effects on G1 to S phase progression in human cancer cells, influencing the levels of cyclin D1 and phosphorylation of Rb (Cell Cycle, 2007). However, other evidence points to the role of 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 TNFa stimulation. More recent proteomic results using mass spectrometry of immunoprecipitated complexes revealed that the SAPS subunits bind a family of Ankyrin Repeat Subunits (we named ARS) that are functionally equivalent to the SAPS themselves in siRNA knockdown assays (see Figure, from Biochemistry 2008). Thus, we now propose that PP6 is a trimeric enzyme, composed of ARS, SAPS and a catalytic subunit.
Our hypothesis is that PP6 and PP2A act to dephosphorylate kinases to modulate cellular responses to Toll-Like Receptors (TLRs) activated during infections and to production of inflammatory cytokines such as Il-1b and TNFa. Current work is pursuing how PP6 and PP2A phosphatases negatively regulate macrophage responses to infections and cytokines.
Our work is supported by grants from the United States Public Health Service (USPHS) - the National Cancer Institute and National Institute of General Medical Sciences.
Dr. Brautigan purifying phosphatases in coldroom, using column chromatography
Most recent update January, 2009.
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