Prof Geraint Thomas
(1) The role of Arf G-proteins in the control of phospholipid metabolism and signalling.
Arfs (ADP-ribosylation factors) are a family of ras-like GTP-binding proteins with roles in cellular signal transduction, adhesion and cargo trafficking. Recently, we have completed a rigorous study of the selectivity of two classes of downstream Arf effectors phospholipase D1 (PLD1) and phosphatiylinositol 4 phosphate 5-kinases (PtdIns4P 5-kinases) between two different Arf isoforms, Arf1 and 6. Arfs 1 and 6 are the most divergent members of this family both structurally and functionally. Existing data on selectivity was not synoptic, principally because of problems associated with the production of recombinant enzymes or Arfs or significant differences in the assay conditions. We have concluded that Arf1/PLD1 is a strongly preferred coupling where as PtdIns4P 5-kinase are entirely promiscuous. We have also established that the likely mode of activation of PtdIns4P 5-kinases by Arfs is by membrane recruitment from the cell cytosol so increasing the apparent substrate on-rate, rather than through significant changes in the kinases specific activity. Simulation studies indicate profound consequences will arises from this Arf isoform selectivity in the coupled, cooperative behaviour of Arf/PLD1/ PtdIns4P 5-kinase triples.
In further work we have concentrated on the specific Arf1/PLD1 interaction and studied selectivity for coupled Arf1/PLD1 amongst upstream guanine nucleotide exchange factors (GEFS) of the ARNO/Cytohesin/Grp1 family. We have established that structural differences between The GEF proteins in this family are redundant in the regulation of PLD1. More recently we have reconstituted the regulation of the Cytohesin1/ARF1/PLD1 triple by polyphosphoinositides. Surprisingly the system responds best to PtdIns(3,4,5)P3, indicating either key roles for still unidentified PtdIns(3,4,5)P3-binding proteins or that this specificity is an emergent property of this system.
(2) The control of polyphosphoinositide metabolism.
Many cell signalling events induce the hydrolysis of phosphatidyinositol 4,5-bisphosphate [PtdIns(4,5)P2] by phospholipase C and the accumulation of the products inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and 1,2-diacylglycerol (DAG). Indeed, many times more PtdIns(4,5)P2 is broken down than is initially present. Consequently, the control of the resynthesis of PtdIns(4,5)P2 by PtdIns4P 5-kinases to meet this increased flux is essential. We have now established that DAG is a direct activator of all known active isoforms of PtdIns4P 5-kinases. This is the shortest and most direct positive feedback loop (a single arc in any model network) to the control of PtdIns(4,5)P2 resynthesis yet discovered and as such is likely to be both the most stable and fastest mechanism of control. Simulation studies incorporating this new regulatory arc model experimentally determined PtdIns(4,5)P2 concentration changes nearly identically.
(3) Control of constitutive protein secretion from the liver.
The biggest endocrine protein secretory event in the body of mature mammals is the continuous secretion of albumin in to the blood. For many years this process was not thought to be regulated but to proceed by a default pathway described as constitutive secretion. In the constitutive model the direction (exit face from the hepatocytes) but not rate of secretion is thought to be controlled. Recently we have shown that protein tyrosine kinases and phosphatases play an essential permissive role in this process and that their inhibition leads to blockade of albumin secretion at the level of Trans Golgi Network (TGN). This is the first demonstration of any role for these key signalling proteins in any constitutive secretory process.
Cancer biomarker discovery
Cell signalling through phospholipases, kinases and G-proteins during differentiation and secretion.
Dr John Timms; Prof Ivan Gout; Prof Simon Gayther; Dr Stephen Pereira; Prof David Linch; Prof Ian Jacobs; Prof Usha Menon
Prof Anne Ridley; Prof John Neoptolemos; Dr Eithne Costello; Dr Hong-Lin Chan; Prof Rainer Cramer; Dr Dolores Pérez-Sala; Dr Pedro Cutillas
- Fourniol FJ, Sindelar CV, Amigues B, Clare DK, Thomas G, Perderiset M, Francis F, Houdusse A, Moores CA (2010). Template-free 13-protofilament microtubule-MAP assembly visualized at 8 Å resolution.. Journal of Cell Biology, 191(3), 463 - 470. doi:10.1083/jcb.201007081
- Thomas GM (2010). Modelling the effects of inhibitors of guanine nucleotide synthesis: implications for studies of cellular differentiation pathways.. Biochem Soc Trans, 38(5), 1314 - 1318. doi:10.1042/BST0381314
- Webb RJ, Tinworth L, Thomas GM, Zaccolo M, Carroll J (2008). Developmentally acquired PKA localisation in mouse oocytes and embryos. Developmental Biology, 317(1), 36 - 45. doi:10.1016/j.ydbio.2008.01.045
- Perez-Mansilla B, Ha VL, Justin N, Wilkins AJ, Carpenter CL, Thomas GM (2006). The differential regulation of phosphatidylinositol 4-phosphate 5-kinases and phospholipase D1 by ADP-ribosylation factors 1 and 6.. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 1761(12), 1429 - 1442. doi:10.1016/j.bbalip.2006.09.006
- Judah JD, Thomas GMH (2006). Two distinct chloride ion requirements in the constitutive protein secretory pathway.. European Journal of Cell Biology, 85, 825 - 836. doi:10.1016/j.ejcb.2006.03.005
- Devader C, Webb RJ, Thomas GMH, Dale L (2006). Xenopus apyrase (xapy), a secreted nucleotidase that is expressed during early development. Gene, 367, 135 - 141. doi:10.1016/j.gene.2005.10.014
- Kramer I, Thomas GMH (2006). Teaching Signal Transduction. CBE Life Sciences Education, 5(1), 19 - 26. doi:10.1187/cbe.05-11-0127
- Webb RJ, Judah JD, Lo LC, Thomas GM (2005). Constitutive secretion of serum albumin requires reversible protein tyrosine phosphorylation events in the trans-Golgi. American Journal of Physiology - Cell Physiology, 289(3), C748 - C756. doi:10.1152/ajpcell.00019.2005
- Haynes LP, Thomas GM, Burgoyne RD (2005). Interaction of neuronal calcium sensor-1 and ADP-ribosylation factor 1 allows bidirectional control of phosphatidylinositol 4-kinase beta and trans-Golgi network-plasma membrane traffic. Journal of Biological Chemistry, 280, 6047 - 6054. doi:10.1074/jbc.M413090200
- Ha VL, Thomas GMH, Stauffer S, Randazzo PA (2005). Preparation of Myristoylated Arf1 and Arf6. Methods in Enzymology, 404, 164 - 174.
- Judah JD, Webb RJ, Knezevic A, Thomas GMH (2003). Inhibition of either protein tyrosine kinases or phosphatases strongly inhibits the secretion but not the synthesis of serum albumin by rat hepatocytes.. J. Physiol., 547(P), - .
- Pinxteren JA, Alb JG, Rostand K, Tatham PE, Thomas GM, Bankaitis VA (2001). Mast cells derived from PITP alpha (-/-) ES cells exocytose happily. MOL BIOL CELL, 12, 474A - 474A.
- Pinxteren JA, Gomperts BD, Rogers D, Phillips SE, Tatham PE, Thomas GM (2001). Phosphatidylinositol transfer proteins and protein kinase C make separate but non-interacting contributions to the phosphorylation state necessary for secretory competence in rat mast cells. Biochemical Journal, 356(1), 287 - 296. doi:10.1042/0264-6021:3560287
- Pinxteren JA, Hammond GR, Thomas GMH (2000). Activation of phosphatidylinositol 4-phosphate 5-kinases by phosphatidylinositol transfer proteins and their contribution to secretory competence in rat mast cells.
- Pinxteren JA, Hammond GR, Thomas GMH (2000). Phosphatidylinositol transfer protein and protein kinase C make separate but non-interacting contributions to the phosphorylation state necessary for secretory competence in rat mast cells.
- Thomas GM, Pinxteren JA (2000). Phosphatidylinositol transfer proteins: one big happy family or strangers with the same name?. Molecular Cell Biology Research Communications, 4(1), 1 - 9.
- Chubb JR, Wilkins A, Thomas GM, Insall RH (2000). The Dictyostelium RasS protein is required for macropinocytosis, phagocytosis and the control of cell movement . Journal of Cell Science, 113(4), 709 - 719.
- Glenn DE, Thomas GMH, O'Sullivan AJ, Burgoyne RD (1998). Examination of the role of ADP-ribosylation factor and phospholipase D activation in regulated exocytosis in chromaffin and PC12 cells. Journal of Neurochemistry, 71, 2023 - 2033.
- Fensome A, Cunningham E, Prosser S, Tan SK, Swigart P, Thomas G, Hsuan J, Cockcroft S (1996). ARF and PITP restore GTP gamma S-stimulated protein secretion from cytosol-depleted HL60 cells by promoting PIP2 synthesis. Current Biology, 6(6), 730 - 738.
- Kauffmann-Zeh A, Thomas GM, Ball A, Prosser S, Cunningham E, Cockcroft S, Hsuan JJ (1995). Requirement for phosphatidylinositol transfer protein in epidermal growth factor signaling. Science, 268(5214), 1188 - 1190.
- Cunningham E, Thomas GM, Ball A, Hiles I, Cockcroft S (1995). Phosphatidylinositol transfer protein dictates the rate of inositol trisphosphate production by promoting the synthesis of PIP2.. Curr Biol, 5(7), 775 - 783.
- Greasley SE, Jhoti H, Teahan C, Solari R, Fensome A, Thomas GMH, Cockcroft S, Bax B (1995). The structure of rat ADP-ribosylation factor-1 (ARF-1) complexed to GDP determined from two different crystal forms.. Nature Structural Biology, 2, 797 - 806. doi:10.1038/nsb0995-797
- Thomas GM, Cunningham E, Cockcroft S (1994). Purification of phosphatidylinositol transfer protein from brain cytosol for reconstituting G-protein-regulated phosphoinositide-specific phospholipase C-beta isozymes.. Methods Enzymol, 238, 168 - 181.
- Thomas GM, Fensome A, Whatmore J, Geny B, Cockcroft S (1994). The regulation of Phospholipase D by ARFs.. Journal of Neurochemistry, 63(Suppl.1), - .
- Greasley S, Jhoti H, Fensome AC, Cockcroft S, Thomas GMH, Bax B (1994). Crystallization and preliminary X-ray diffraction studies on ADP-ribosylation factor 1.. Journal of Molecular Biology, 244, 651 - 653. doi:10.1006/jmbi.1994.1759,
- Cockcroft S, Thomas GM, Cunningham E, Ball A (1994). Use of cytosol-depleted HL-60 cells for reconstitution studies of G-protein-regulated phosphoinositide-specific phospholipase C-beta isozymes.. Methods Enzymol, 238, 154 - 168.
- Cockcroft S, Thomas GM, Fensome A, Geny B, Cunningham E, Gout I, Hiles I, Totty NF, Truong O, Hsuan JJ (1994). Phospholipase D: a downstream effector of ARF in granulocytes.. Science, 263(5146), 523 - 526.
- Thomas GM, Cunningham E, Ball A, Fensome A, Cockcroft S (1994). An Essential role for phosphatidylinositol transfer protein in inositol lipid signalling.. Journal of Cellular Biochemistry, (Suppl.18D), - .
- Thomas GM, Cunningham E, Fensome A, Ball A, Totty NF, Truong O, Hsuan JJ, Cockcroft S (1993). An essential role for phosphatidylinositol transfer protein in phospholipase C-mediated inositol lipid signaling.. Cell, 74(5), 919 - 928.
- Cockcroft S, Thomas GM (1992). Inositol-lipid-specific phospholipase C isoenzymes and their differential regulation by receptors.. Biochem J, 288 ( Pt 1), 1 - 14.
- Thomas GM, Geny B, Cockcroft S (1991). Identification of a novel cytosolic poly-phosphoinositide-specific phospholipase C (PLC-86) as the major G-protein-regulated enzyme.. EMBO J, 10(9), 2507 - 2512.
- Cockcroft S, Geny B, Thomas GM (1991). Regulation of cytosolic phosphoinositide-phospholipase C by G-protein, Gp.. Biochem Soc Trans, 19(2), 299 - 302.
- Birch NJ, Thomas GMH, Hughes MS (1991). Cell signalling: Experimental Strategies.
- Thomas GM, Olufunwa RI (1988). Lithium: Inorganic Pharmacology and Psychiatric Use.
- Partridge S, Hughes MS, Thomas GMH , Birch NJ (1988). Lithium transport in erythrocytes.. Biochemical Society Transactions, 16, 205 - 206.
- Hughes MS, Thomas GMH, Partridge S, Birch NJ (1988). An investigation into the use of dysprosium shift reagent in the nuclear magnetic resonance spectroscopy of biological systems.. Biochemical Society Transactions, 16, 207 - 208.
- Birch NJ, Hughes, MS , Thomas GMH , Partridge, S (1986). Lithium and Magnesium: Inorganic pharmacology.. Magnesium Bulletin, 8, 145 - 147.
- Thomas GM, Hughes MS, Partridge S, Olufunwa RI, Marr G, Birch NJ (). N.m.r. studies of lithium transport in isolated rat hepatocytes.. Biochemical Society Transactions, 16, 206 - 206.