Intracellular signalling and cancer

Professor C.A. Mitchell

Professor Christina Mitchell: Signal Termination by Lipid Phosphatases

The proto-oncogene PI 3-kinase produces critical signalling molecules including PtdIns(3,4,5)P3, and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). These signalling molecules bind the pleckstrin homology (PH) domain of the proto-oncogene serine/threonine kinase, Akt, which inhibits apoptosis; proteins that regulate Rho and Rac and thereby actin-dependent extension of lamellipodia, and cell migration, ARF GEFs/GAPs and thereby vesicular trafficking. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are virtually undetectable in unstimulated cells, however, these signaling molecules are rapidly synthesized in response to extracellular agonist stimuli. Amplication of these lipid signalling molecules is detected in many cancers. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are rapidly metabolized by specific lipid phosphatases that dephosphorylate at the 5-, 4- or 3-position phosphate. The phosphatase and tensin homologue deleted on chromosome ten (PTEN) is a tumour suppressor gene product that acts as a lipid 3-phosphatase.

The 5-phosphatases are an enzyme family that hydrolyse the 5-position phosphate from the inositol ring of inositol phosphates and/or phosphoinositides such as PtdIns(3,4,5)P3 and PtdIns(4,5)P2 (see Figure above). Ten mammalian enzymes have been cloned and characterized and four yeast homologues have been identified in Saccharomyces cerevisiae. The mammalian 5-phosphatases SHIP-1, SHIP-2, Synaptojanin and Lowe's 5-phosphatase demonstrate significant cellular and physiological function as shown by gene targetted knockout studies. These 5-phosphatases regulate such pathophysiological processes as leukemia, insulin signalling, neuronal signalling and vesicular trafficking respectively.

Our laboratory has recently cloned three new mammalian 5-phosphatases that play important roles in cancer biology, cell proliferation and insulin signalling (see Figure below). We have also undertaken yeast two hybrid strategy to identify the signalling pathways in which these 5-phosphatases interact, and determined using expression of these recombinant proteins with green fluorescent protein, the intracellular location of each 5-phosphatase.

Project Areas focus on the regulation of cancer cell growth/death and differentiation. Studies are performed using a significant number of molecular, cell biology and protein chemistry techniques. We are also developing both transgenic and gene-targetted deletion in mice (knockout mice) to use a models to investigate the role the lipid phosphatases play in cell differentiation and cancer.

Project Areas

1. The role of the 72 kDa 5-phosphatase in cancer cell proliferation

We have recently cloned a 72 kDa 5-phosphatase which localizes to the cytosolic face of the Golgi and the cell cytosol. The 72 kDa enzyme is a potent inhibitor of cell death via regulation of the proto-oncogene Akt. Overexpression of the 72 kDa 5-phosphatase results in rapid cell death. We have also recently demonstrated in highly differentiated cells the 72 kDa 5-phosphatase may also regulate secretion. This project aims to investigate the role the 72 kDa enzyme plays in regulating cell growth by identifying and characterising other signalling proteins which complex with the 5-phosphatase using a variety of molecular, genetic and cell biology techniques including yeast two hybrid analysis, generation of stable cell lines, and analysis of transgenic animals overexpressing the 72 kDa 5-phosphatase.

2: Characterization of SKIP and binding partners that inhibit apoptosis.

The inositol polyphosphate 5-phosphatase SKIP terminates PI 3-kinase signals by hydrolysing PtdIns(3,4,5)P3. We have recently identified using yeast two hybrid screening that SKIP forms a complex with a signalling protein that potently inhibits cell death. We propose the 5-phosphatase SKIP terminates PI 3-kinase signals via complex with specific anti- apoptotic signalling proteins. This project aims to use molecular and cell biology techniques to characterise the cell death signalling pathways regulated by this complex by generating stable cell lines and transgenic animals that overexpress wild type or dominant negative SKIP and its anti-apoptotic binding partners. We will investigate the interaction between these proteins using both in vitro and in vivo analysis, characterise the down-stream apoptotic signalling pathways regulated by SKIP.

3. Characterization of the 107 kDa 5-phosphatase

The 107 kDa 5-phosphatase is a recently identified member of the 5-phosphatase enzyme family but very little is known about this enzyme's cellular function. The enzyme has extensive N-terminal and C-terminal proline rich domains indicating it may form complexes with other signalling proteins. This project will use yeast two hybrid analysis to identify novel proteins which complex with this novel 5-phosphatase and determine their cellular function.

Prof Christina Mitchell and Dr Susan Brown SLIM PROTEINS: ROLE IN CARDIAC AND SKELETAL MUSCLE DEVELOPMENT AND DISEASE.

Cardiac failure due to previous heart attacks or inherited cardiomyopathy is one of the major causes of death and disability in our community. Large scale genetic screens have identified the gene SLIM1 as one of the most significantly downregulated genes in dilated cardiomyopathy, a weakening and thinning of the heart muscle which develops following severe heart attacks. Conversely, SLIM1 levels increase significantly in cardiac hypertrophy (heart muscle thickening). Cardiac hypertrophy may develop either as a consequence of longstanding hypertension, or due to an inherited defect of heart muscle proteins and is the major cause of sudden, unexplained death in young adults.

Our laboratory is examining the role of the SLIM proteins, which stands for skeletal muscle LIM proteins, in cardiac and skeletal muscle. The SLIM proteins are each comprised of four and a half LIM domains. LIM domains are double zinc finger structures, which bind proteins. LIM domain containing proteins have critical roles in tissue differentiation and have been associated with human diseases, including T-cell leukaemia and a congenital form of mental retardation.

SLIM1/FHL1 is expressed in both skeletal and cardiac muscle. We have demonstrated that SLIM1 is expressed early in the embryo in the developing heart and in skeletal muscle precursors. Furthermore, overexpression of SLIM1 promotes the development of skeletal myotube hypertrophy in vitro. We have recently determined the localization of SLIM in normal and diseased hearts and demonstrated a specific pathology. Collectively these studies indicate SLIM1 regulates cardiac myocyte function in normal and diseased hearts.

We are currently generating transgenic mice overexpressing SLIM1 specifically in cardiac and skeletal muscle, to determine whether increased SLIM1 levels can cause respectively, cardiac and skeletal muscle hypertrophy in vivo.

Project Areas:

1. Demonstration of the precise role SLIM1 performs in normal muscle and in the development of cardiomyopathy, will be achieved by identifying the muscle cytoskeletal and signaling proteins it interacts with via its LIM domains. A yeast two-hybrid screen to identify SLIM1 protein binding partners in skeletal and cardiac muscle has already identified a number of muscle proteins, which complex with SLIM1. Potential binding partners of SLIM1 will be confirmed in in vitro and in vivo studies, and functional consequences of this interaction on the development of muscle hypertrophy assessed.

2. Analysis of transgenic mice overexpressing SLIM1 in cardiac or skeletal muscle, via a number of molecular and imaging techniques to determine the effect on muscle function and structure.

Recent Selected Publications

1. Dyson JN, O'Malley CJ, Becanovic J, Munday AD, Berndt MC, Coghill ID, Nandurkar HH, Ooms LM and Mitchell CA.  (2001) The SH2 containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin. J Cell Biol 155, 1065-1079 IF13

2. Dyson JM, Munday AD, Kong AM, Huysmans RD, Matzaris M, Layton MJ, Nandurkar HH, Berndt MC and Mitchell CA.  (2003) SHIP-2 forms a tetrameric complex with filamin, actin, and GPIb-IX-V.  Localization of SHIP-2 to the activated platelet actin cytoskeleton. Blood 102, 940-948  IF 10

3. Nandurkar HH, Layton M, Laporte J, Selan C, Corcoran L, Caldwell KK, Mochizuki Y, Majerus PW and Mitchell CA.  (2003) Identification of myotubulin as the lipid phosphatase catalytic subunit associated with the 3-phosphatase adpater protein, 3-PAP. Proc Natl Acad Sci USA 100, 8660-8665 IF 11

4. McGrath MJ, Mitchell CA, Coghill ID, Robinson PA and Brown S.  (2003) Skeletal muscle LIM protein (SLIM1/FHL1) induces alpha 5 beta 1- integrin dependent myocyte elongation Am J Physiol Cell Physiol 285, C1513-26

5. Ooms LM, Fedele CG, Ivetac I, Astle MV, Pearson RB, Layton MJ, Forrai A,  Nandurkar HH and Mitchell CA.  (2006) The inositol polyphosphate 5-phosphatase, PIPP, is a novel regulator of phosphoinositide 3-kinase-dependent neurite elongation. Mol Biol Cell 17, 607-22  IF 7.5

6. McGrath MJ, Cottle DL, Nguyen MA, Dyson JM, Coghill ID, Robinson PA, Holdsworth M, Cowling BS, Hardeman EC, Mitchell CA and Brown S.  (2006) Four and half LIM protein 1 binds myosin-binding protein C and regulates myosin filament assembly and sarcomere formation. J Biol Chem 17, 7666-83  IF 6

7. Kong AM, Horan KA, Sriratana A, Bailey CG, Collyer LJ, Nandurkar HH, Shisheva A, Layton M, Rasko JEJ, Rowe T and Mitchell CA.  (2006) Phosphatidylinositol (3) phosphate is generated at the plasma membrane by inositol polyphosphate 5-phosphatase hydrolysis of phosphatidylinositol (3,5) bisphosphate. Mol Cell Biol 26,6065-81 IF7.5

8. Cottle D, McGrath M, Cowling B, Brown S and Mitchell CA.  (2007) FHL3 binds MyoD and negatively regulates myotube formation. J Cell Sci 120, 1423-35. IF 7

9. Wiradjaja F, Ooms LM, Tahirovic S, Kuhne D, Devenish RJ, Munn AL, Piper RC, Mayinger P and Mitchell CA.  (2007) Inactivation of the phosphoinositide phosphatases Sac1p and Inp54p leads to accumulation of PtdIns(4,5)P2 on vacuole membranes and vacuolar fusion defects. J Biol Chem 282, 16295-307.  IF6

10. Horan KA, Watanabe KI, Kong AM, Bailey CG, Rasko JE, Sasaki T and Mitchell       CA.  (2007) Regulation of Fc{gamma}R-stimulated phagocytosis by the 72 kDa inositol polyphosphate 5-phosphatase:  SHIP1, but not the 72 kDa 5-phosphatase, regulates complement receptor-3-mediated phagocytosis, by differential recruitment of these 5-phosphatases to the phagocytic cup.  Blood. 110, 4480-91 (IF 10.3)

11. Waters JE, Astle MV, Ooms LM, Balamatsias D, Gurung R, and Mitchell CA. (2008) P-Rex1, a multidomain protein that regulates neurite differentiation. J Cell Sci 121, 2892-903 (IF 7).

12. Cowling BS, Nguyen M-A, McGrath MJ, Cottle DL, Kee AJ, Brown S, Schessl J, Zou Y, Joya J, Bönnemann CG, Hardeman EC and Mitchell CA.  Identification of FHL1 as a novel regulator of skeletal muscle mass: implications for human myopathy. J Cell Biol (impact factor 11) (in press).

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