Centre for Cancer Biology
You are here: Molecular Regulation Laboratory

General Enquiries

Ms Anna Nitschke
PA to Prof. Angel Lopez

Centre for Cancer Biology
SA Pathology
PO Box 14 Rundle Mall
Adelaide SA 5000
AUSTRALIA

Tel: 61 8  8222 3422
Fax:61 8  8232 4092


Email:

Anna.Nitschke@health.sa.gov.au




  Molecular Regulation Laboratory

Head

Prof Sharad Kumar

Address

 

Centre for Cancer Biology
SA Pathology
Frome Road,
Adelaide SA 5000, Australia
Phone +61 8 8222 3738
Fax +61 8 8222 3162
Email sharad.kumar@health.sa.gov.au

Affiliations:

NHMRC Senior Principal Research Fellow
Professor (affiliate), Department of Medicine, University of Adelaide


Qualifications:

MSc, PhD


Lab Members

Postdoctoral Research Scientists Telephone Email
Balazs Bajka 8222 3604 balazs.bajka@imvs.sa.gov.au
Natasha Boase 8222 3604 natasha.boase@imvs.sa.gov.au
Hazel Dalton 8222 3604 hazel.dalton@imvs.sa.gov.au
Donna Denton 8222 3604 donna.denton@imvs.sa.gov.au
Loretta Dorstyn 8222 3604 loretta.dorstyn@imvs.sa.gov.au
Saman Ebrahimi 8222 3604 saman.ebrahimi@imvs.sa.gov.au
Natalie Foot 8222 3604 natalie.foot@imvs.sa.gov.au
Sonia Shalini 8222 3604 sonia.shalini@imvs.sa.gov.au
   
Research Assistants    
Kristen Ho 8222 3604 kristen.ho@imvs.sa.gov.au
Yew Ann Leong 8222 3604 yewann.leong@imvs.sa.gov.au
Kathryn Mills 8222 3604 kathryn.mills@imvs.sa.gov.au
Scott Townley 8222 3604 scott.townley@imvs.sa.gov.au
   
PhD Students    
Jantina Manning 8222 3604 jantina.manning@imvs.sa.gov.au
   
Honor Students    
Joey Puccini 8222 3604 joey.puccini@imvs.sa.gov.au

 


From left, Back Row: Yew Ann Leong, Balazs Bajka, Loretta Dorstyn, Hazel Dalton, Natasha Boase, Saman Ebrahimi, Donna Denton, Katherine Adriaanse,Front Row: Sonia Shalini, Jantina Manning, Sharad Kumar, Natalie Foot, Joey Puccini, Kristen Ho

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Research Focus

The Molecular Regulation laboratory undertakes research in the general area of cell and molecular biology of disease. The two major interests of the Molecular Regulation laboratory are (1) the study of programmed cell death (apoptosis) of normal and cancer cells and (2) understanding the regulation of ion channels and transporters by ubiquitination.

Apoptosis plays a fundamental role in cell and tissue homeostasis and its misregulation results in a variety of human diseases including many types of cancer. As apoptosis is a major mechanism for deleting harmful cells from the body, deciphering the mechanisms of apoptosis is essential for understanding disease processes and to design effective treatment strategies for diseases which arise due to inappropriate apoptosis. We are studying the function and regulation of caspases, a group of enzymes that act as effectors of apoptosis, in mediating apoptosis and in killing cancer cells. We use a range of biochemical, cellular and whole animal approaches for these studies. We also use gene knock-out mice and mouse models of tumour development to study the role of caspases in cancer.

Ubiquitin-mediated protein modification plays an essential role in cellular regulation during embryonic development, transcription and the cell cycle. Recent studies suggest that ubiquitination is a major regulator of many ion channels, receptors and transporters. We are studying the function of a group of ubiquitin-protein ligating enzymes (Nedd4-like proteins), which are key in defining substrate specificity of the ubiquitin system. We are using a variety of molecular, cellular and gene knockout approaches to study the physiological functions of these enzymes and establish their role in human diseases, such as haemochromatosis, anaemia and hypertension.

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Research Projects

1. Caspase function in apoptosis and cancer cell death

mouse Loretta Dorstyn, Sonia Shalini, Balazs Bajka and Joey Puccini

Caspases are cysteine proteases that act as executioners of apoptosis. Having cloned one of the first caspases (caspase-2), our laboratory has an ongoing program in understanding caspase biology, regulation and function. Our current focus is to delineate the in vivo function of caspase-2. Accumulated data support a critical function for caspase-2 as an initiator caspase which links death signals to mitochondrial outer membrane permeabilization (MOMP). We have found that while mouse embryonic fibroblasts (MEFs) from caspase-2 null mice are normally sensitive to a number of chemotherapeutic drugs, they show significant resistance to killing by drugs that are known to induce apoptosis by disrupting the cytoskeleton. Reduced caspase-2 expression is often associated with many cancers and low caspase-2 levels have been correlated with drug-resistance. Thus it is likely that caspase-2 is required for apoptosis under certain pathological conditions, such as cancer. We are using caspase-2 knockout mice and mouse models of cancer to understand whether the loss of caspase-2 contributes to tumorigenesis. Indeed, using the mouse Em-Myc lymphoma model we found that the loss of even a single allele of caspase-2 resulted in accelerated tumourigenesis, and this was further enhanced in caspase-2-/- mice. In addition, caspase-2 has been implicated in DNA repair pathways, and we are further investigating this. Finally, a loss of caspase-2 has been shown to promote aging. We are investigating the mechanism by which caspase-2 prevents premature aging in mice.


fig 1
The lack of caspase-2 accelerates tumour formation in male athymic nude mice. Mice injected with E1A/Ras-transformed caspase-2−/− MEFs (lower RHS panels showing mice at day 10) develop more aggressive tumours than those injected with saline or E1A/Ras-transformed caspase-2+/+ MEFs.

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2. Drosophila as a model to study developmental cell death


Donna Denton and Kathryn Mills


Many of the programmed cell death (PCD) components and pathways found in mammals are conserved in Drosophila, thus it is useful model system to study cell death regulation during development. The ease of genetic manipulation makes Drosophila an ideal system to study programmed cell death in vivo. There are seven caspases in Drosophila including DCP-1, DREDD/DCP-2, DRICE, DRONC, DECAY, STRICA and DAMM. Four of these, DRONC, STRICA, DAMM and DECAY were identified in our laboratory. We have found that the caspase DRONC (Drosophila Nedd-2-like caspase), along with its adaptor protein ARK, are essential for developmental PCD in Drosophila. We are using genetic and biochemical approaches to further understand the role of caspases during developmental processes, including their potential function in autophagic cell death in larval tissues.

fig 1

Using Drosophila to study cell death. Analysis of apoptosis in the adult Drosophila compound eye and wing (A-D) and analysis of apoptotic cells in the larval midgut by TUNEL staining (E).

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3. Hormone regulation of transcription and characterisation of novel histone modifying enzymes

Saman Ebrahimi, Kathryn Mills, Donna Denton

The members of the nuclear hormone receptor (NHR) family bind their cognate ligands to regulate diverse physiological processes such as proliferation, differentiation and PCD by modulating transcription of a distinct array of genes. NHRs act via recruiting coactivators that are capable of modifying and remodelling chromatin structure. One of the projects in our lab is to understand the molecular mechanisms involved in hormone-mediated PCD by analysing the transcriptional regulation of the genes involved in apoptosis. We use both Drosophila and mammalian cells as experimental systems to study the function of NHRs in PCD. Ultimately all developmental signals lead to histone modifications such as methylation, acetylation, ubiquitination and phosphorylation, resulting in chromatin remodelling and gene activation and/or repression. In diseases such as cancer, enzymes that bring about these modifications are often deregulated. We are identifying and cloning enzymes/proteins that regulate methylation/demethylation of histones in Drosophila and mammalian cells. With the recent discovery of the histone demethylase protein family, some of our current work involves the analysis of new histone demethylases involved in nuclear hormone mediated transcription, proliferation, differentiation and PCD in Drosophila. We are also studying potential roles of these epigenetic modifiers in cancer. We use cellular, molecular, proteomic and genetic approaches to study the functions of these enzymes.


fig 1
Ecdysone regulates expression of cell death genes during Drosophila metamorphosis.

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4. Nedd4 proteins in physiology and disease

Natasha Boase, Scott Townley and Jantina Manning

Collaborators: David Cook (University of Sydney), Philip Poronnik (University of Queensland), Grigori Rychkov (University of Adelaide), Baoli Yang (University of Iowa) and Roger Daly (Garvan Institute)

Aberrations in the ubiquitin system underpin the pathogenesis of many diseases including malignancies, neurodegenerative disorders and channelopathies. Ubiquitin-protein ligases (E3s) determine the substrate specificity of the ubiquitination process. The Nedd4 family of E3s is evolutionarily conserved and required for the ubiquitination of numerous cellular targets involved in processes such as transcription, stability and trafficking of plasma membrane proteins, and the degradation of misfolded proteins. Nedd4 is a gene initially identified in our laboratory. Members of the Nedd4-family can ubiquitinate a range of membrane proteins, resulting in their internalisation and degradation. We have shown that Nedd4, and the closely related protein Nedd4-2, interacts with and ubiquitinates the epithelial sodium channel (ENaC). ENaC is required for sodium absorption across a range of epithelial tissues such as the lungs, colon and kidney and is an important regulator of blood sodium concentration. Ubiquitination of ENaC by Nedd4 and Nedd4-2 leads to its internalisation and degradation. Defects in this process disrupt sodium homeostasis and can cause hypertension. Our current focus is to characterise the mechanisms of regulation of ENaC and other ion channels (such as voltage-gated sodium channels) by Nedd4 and Nedd4-2.


fig 1 Regulation of ENaC by Nedd4-2. When intracellular Na+ levels are low, various hormones activate Sgk1, Akt or PKA, which phosphorylate Nedd4-2. 14-3-3 binds to phosphorylated Nedd4-2 to prevent its interaction with ENaC, resulting in an increase in the levels of ENaC at the plasma membrane. When intracellular Na+ is high, Nedd4-2 ubiquitinates ENaC, which leads to its internalisation and degradation.

In a collaborative study we have recently found that the loss of Nedd4 in mice results in reduced IGF-1 and insulin signalling, reduced growth and neonatal lethality. Nedd4-deficient cells show reduced mitogenic activity. This appears to be due to increased levels of the adaptor protein Grb10 resulting in IGF-1R mislocalization and inhibition of IGF-1 and insulin signalling. We are now studying the mechanism of Grb10 regulation by Nedd4.

There is evidence to suggest that Nedd4 has additional cellular targets. Thus, we are analysing additional phenotypes that may be associated with the knockout of Nedd4.

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5. Ndfips as regulators of Nedd4 family members

Hazel Dalton, Loretta Dorstyn, Natalie Foot, Kristen Ho and Yew Ann Leong

Collaborators: Seong Seng Tan (Howard Florey Institute) and Baoli Yang (University of Iowa)

We have identified a number of Nedd4-interacting proteins. Two such proteins are Ndfip1 and Ndfip2, which display Golgi and endosomal localisation, suggestive of a role in protein trafficking. Based on our data, Ndfip1 and Ndfip2 are predicted to function as adaptor proteins that recruit Nedd4 family E3s to their substrates to provide specificity and regulatory complexity to the ubiquitination system.


Our recent work suggests that Ndfips regulate the divalent metal ion transporter DMT1, the primary non-heme iron transporter in mammals. DMT1 interacts with both Ndfip1 and Ndfip2, and this promotes DMT1 ubiquitination and degradation by the Nedd4-family ubiquitin ligase, WWP2 (see Figure). Consistent with these observations Ndfip1-/- mice show increased hepatic iron deposition, indicating an essential function of Ndfip1 in iron homeostasis. Given that misregulation of DMT1 is implicated in a number of human diseases, Ndfips and WWP2 could be potential targets for therapeutic intervention to control iron uptake and/or metabolism. Furthermore, mutations and variations in WWP2 and Ndfip genes may result in diseases of iron metabolism. Our current focus is to further characterise WWP2 and Ndfips to provide additional understanding of this novel mechanism of regulating iron transport.

fig 1 Using Ndfip1-/- mice to show how Ndfip1 is involved in iron homeostasis. Ndfip1-/- mice show increased levels of DMT1 in the liver (A-B) which is associated with iron loading (C). The ion transport activity in primary hepatocytes is also increased in Ndfip1-/- mice compared to wild type littermates (D).

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Selected Recent Publications

2009

Hiwase DK, White DL, Powell JA, Saunders VA, Zrim SA, Frede AK, Guthridge MA, Lopez AF, D’Andrea RJ, To LB, Melo JV, Kumar S, Hughes TP. Blocking cytokine signaling along with intense Bcr-Abl kinase inhibition induces apoptosis in primary CML progenitors. Leukemia In press.

Dorstyn L, Kumar S (2009) Putative functions of caspase-2. F1000 Biology Reports In press.

Denton D, Shravage B, Simin R, Baehrecke EH, Kumar S. Larval midgut destruction in Drosophila: Not dependent on caspases but suppressed by the loss of autophagy. Autophagy In press.

Kumar S (2009) Caspase 2 in apoptosis, DNA damage response and tumor suppression: enigma no more? Nature Rev. Cancer 9: 897-903.
 
Denton D, Shravage B, Simin R, Mills K, Berry DL, Baehrecke EH, Kumar S (2009) Autophagy, not apoptosis, is essential for midgut cell death in Drosophila. Current Biology 19: 1741-1746.

Yang B, Kumar S (2009) Nedd4 and Nedd4-2: Closely related ubiquitin-protein ligases with distinct physiological functions. Cell Death Differ. In press. (doi:10.1038/cdd.2009.84)

Howitt J, Putz U, Lackovic J, Doan A, Dorstyn L, Cheng H, Yang B, Chan-Ling T, Silke J, Kumar S, Tan S-S (2009) Divalent metal transporter 1 (DMT1) regulation by Ndfip1 prevents metal toxicity in human neurons. Proc. Natl. Acad. Sci. USA 106:15489-15494.

Kumar S, Dorstyn L (2009) Analyzing caspase activation and caspase activity in apoptotic cells. In: Apoptosis Methods and Protocols (eds. Peter Erhardt and Ambrus Toth). Humana Press Inc. NJ, USA. Methods in Molecular Biology 559: 3-17.

Galluzzi L, Aaronson SA, Abrams J, Alnemri ES, Andrews DW, Ashkenazi A, Baehrecke EH, Bazan NG, Blagosklonny MV, Blomgren K, Borner C, Bredesen DE, Brenner C, Castedo M, Cidlowski JA, Ciechanover A, Cohen GM, De Laurenzi V, Maria RD, Deshmukh M, Dynlacht BD, El-Deiry WS, Fulda S, Garrido C, Golstein P, De Maria R, Deshmukh M, Dynlacht BD, El-Deiry WS, Flavell RA, Fulda S, Garrido C, Golstein P, Gougeon M-L, Green DR, Gronemeyer H, Hajnoczky G, Hardwick JM, Hengartner M, Ichijo H, Jäättelä M, Kepp O, Kimchi A, Klionsky DJ, Knight RA, Kornbluth S, Kumar S, Levine B, Lipton SA, Lugli E, Madeo F, Malorni W, Marine J-C W, Martin SJ, Medema JP, Mehlen P, Melino G, Moll UM, Morselli E, Nagata S, Nicholson DW, Nicotera P, Nuñez G, Oren M, Penninger J, Pervaiz S, Peter ME, Piacentini M, Prehn JHM, Puthalakath H, Rabinovich G, Rizzuto R, Rodrigues CMP, Rubinsztein DC, Rudel T, Scorrano L, Simon H-U, Steller H, Tschopp J, Tsujimoto Y, Vandenabeele P, Vitale I, Vousden KH, Youle RJ, Yuan J, Zhivotovsky B, Kroemer G (2009) Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death Differ. 16: 1093–1107.

Kochetkova M, Kumar S, McColl S (2009) The chemokine receptors CXCR4 and CCR7 promote metastasis by preventing anoikis in cancer cells. Cell Death Differ. 16: 664-673.

Ho LH, Taylor R, Cakouros D, Dorstyn L, Bouillet P, Kumar S (2009) A tumor suppressor function for caspase-2. Proc. Natl. Acad. Sci. USA 106: 5336-5341.

Rotin D, Kumar S (2009) Physiological functions of the HECT family of ubiquitin ligases. Nature Rev. Mol. Cell Biol. 10: 398-409.

Lee I-H, Campbell CR, Song S-H, Day ML, Kumar S, Cook DI, Dinudom A (2009) The activity of the epithelial sodium channels is regulated by the caveolin-1 via a Nedd4-2 dependent mechanism. J. Biol. Chem. 284: 12663-12669.

Hiwase DK, White DL, Saunders V, Frede A, To LB, Kumar S, Melo JV, Hughes TP (2009) Short term intense Bcr-Abl kinase inhibition with nilotinib is adequate to trigger cell death in BCR-ABL+ cells. Leukemia 23: 1205-1206.

Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH, Blagosklonny MV, El-Deiry WS, Golstein P, Green DR, Hengartner M, Knight RA, Kumar S, Lipton SA, Malorni W, Nuñez G, Peter ME, Tschopp J, Yuan J, Piacentini M, Zhivotovsky B, Melino G (2009) Classification of cell death: Recommendations of the nomenclature committee on cell death 2009. Cell Death Differ. 16: 3-11.

2008

Dorstyn L, Kumar S (2008) A biochemical analysis of the activation of the Drosophila caspase DRONC. Cell Death Differ. 15: 461-470.

Cao XR, Lill NL, Boase N, Shi PP, Croucher D, Shan H, Qu J, Sweezer EM, Place T, Kirby PA, Daly RJ, Kumar S*, Yang B* (2008) Nedd4 controls animal growth by regulating IGF-1 signaling. Science Signaling 1: ra5. (* joint senior authors).

Ho LH, Dorstyn L, Lambrusco, L, Read SH, Kumar S (2008) Caspase-2 is required for cell death induced by cytoskeletal disruption. Oncogene 27: 3393-3404.

Denton D, Mills K, Kumar S (2008) Methods and protocols for studying cell death in Drosophila. Methods in Enzymology 446: 17-37.

Cakouros D, Mills K, Denton D, Daish T, Paterson A, Kumar S (2008) dLKR/SDH regulates hormone mediated histone arginine methylation and transcription of cell death genes. J. Cell Biol. 182: 481-495.

Manning JA, Colussi PA, Koblar SA, Kumar S (2008) Nedd1 expression as a marker of dynamic centrosomal localization during mouse embryonic development. Histochem. Cell Biol. 129: 751-764.

Hiwase DK, Saunders V, Hewett D, Frede A, Zrim S, Dang P, Eadie L, To LB, Melo J, Kumar S, Hughes TP, White DL (2008) Dasatinib cellular uptake and efflux in CML cells: Therapeutic implications Clin. Cancer Res. 14: 3881-3888.

Schuetz F, Kumar S, Poronnik P, Adams DJ (2008) Regulation of the voltage-gated K+ channels KCNQ2/3 and 3/5 by the serum- and glucocorticoid-regulated kinase (SGK-1). Am. J. Physiol. Cell Physiol. 295: C73-C80.

He Y, Hryciw DH, Carroll ML, Myers SA,Whitbread AK, Kumar S, Poronnik P, Hooper JD (2008) The ubiquitin-protein ligase Nedd4-2 differentially interacts with and regulates members of the Tweety family of chloride ion channels. J. Biol. Chem. 283: 24000-24010.

Foot NJ, Dalton HE, Shearwin-Whyatt LM, Dorstyn L, Tan SS, Yang, B, Kumar S (2008) Regulation of the divalent metal ion transporter DMT1 and iron homeostasis by a ubiquitin-dependent mechanism involving Ndfips and WWP2. Blood 112: 4268-4275.

Putz U, Howitt J, Lackovic J, Foot NJ, Kumar S, Silke J, Tan S-S (2008) Nedd4-family interacting protein 1 (Ndfip1) is required for the exosomal secretion of Nedd4-family proteins. J. Biol. Chem. 283: 32621-32627.

2007

Manning J, Kumar S (2007) NEDD1: Function in microtubule nucleation, spindle assembly and beyond. Int. J. Biochem. Cell Biol. 39: 7-11.

Doumanis J, Dorstyn L, Kumar S (2007) Molecular determinants of the subcellular localization of the Drosophila Bcl-2 homologues DEBCL and BUFFY. Cell Death Differ. 14: 907-915.

Dibbens LM, Ekberg J, Taylor I, Hodgson BL, Conroy S-J, Lensink IL, Kumar S, Zielinski MA, Harkin LA, Sutherland GR, Adams DJ, Berkovic SF, Scheffer IE, Mulley JC, Poronnik P (2007) NEDD4-2 as a candidate susceptibility gene for epileptic photosensitivity. Genes Brain Behav. 6: 750-755.

Kumar S (2007) Caspases and their many biological functions. Cell Death Differ. 14: 1-2.

Ekberg J, Schuetz F, Boase NA, Conroy S-J, Manning J, Kumar S, Poronnik P, Adams DJ (2007) Regulation of the voltage-gated K+ channels KCNQ2/3 and KCNQ3/5 by ubiquitination: Novel role for Nedd4-2. J. Biol. Chem. 282:12135-12142.

Sanchez-Perez A, Kumar S, Cook DI (2007) GRK2 interacts with and phosphorylates Nedd4 and Nedd4-2. Biochem. Biophys. Res. Commun. 359: 611-615.

Lee I-H, Dinudom A, Sanchez-Perez A, Kumar S, Cook DI (2007) Akt mediates the effects of insulin on epithelial sodium channel by inhibiting Nedd4-2. J. Biol. Chem.282: 29866-29873.

Kumar S (2007) Caspase function in programmed cell death. Cell Death Differ. 14: 32-43.

2006

Fotia AB, Cook DI, Kumar S (2006) The ubiquitin-protein ligases Nedd4 and Nedd4-2 show similar ubiquitin-conjugating enzyme specificities. Int. J. Biochem. Cell Biol. 23: 472-479.

Rauh R, Dinudom A, Fotia AB, Paulides M, Kumar S, Korbmacher C, Cook DI (2006) Stimulation of the epithelial sodium channel (ENaC) by the serum- and glucocorticoid-inducible kinase (Sgk) involves the PY-motifs of the channel but is independent of sodium feedback inhibition. Pflug. Arch. Eur. J. Physiol. 452: 290–299.

Shearwin-Whyatt L, Dalton H, Foot N, Kumar S (2006) Regulation of functional diversity within the Nedd4 family by accessory and adaptor proteins. BioEssays 28: 617-628.

Dorstyn L, Kumar S (2006) A cytochrome c-free fly apoptosome. Cell Death Differ. 13: 1049- 1051.

Mills K, Daish, T, Harvey KF, Pfleger CM, Hariharan IK, Kumar S (2006) The Drosophila melanogaster Apaf-1 homologue ARK is required for most, but not all, programmed cell death. J. Cell Biol. 172: 809-815.

Sang Q, Kim MH, Kumar S, Bye N, Morganti-Kossman MC, Gunnersen J, Fuller S, Howitt J, Hyde L, Beissbarth T, Scott HS, Silke J, Tan SS (2006) Nedd4-WW domain-binding protein 5 (Ndfip1) is associated with neuronal survival after acute cortical brain injury. J. Neurosci. 26: 7234-7244.

Daish T, Kumar S (2006) Biology of caspases. In: Apoptosis, Cell Signaling and Human Diseases: Molecular Mechanisms Vol 2 (R. Srivastava, Editor). The Humana Press, Inc. pp 347-363.

2005

Mills K, Daish T, Kumar S (2005) The function of the Drosophila caspase DRONC in cell death and development. Cell Cycle 4:744-746.

Kilpatrick ZE, Cakouros D, Kumar S (2005) Ecdysone-mediated upregulation of the effector caspase DRICE is required for hormone-dependent apoptosis in Drosophila cells. J. Biol. Chem. 280: 11981- 11986.

2004

Daish T, Cakouros D, Kumar S (2003) Distinct promoter regions regulate spatial and temporal expression of the Drosophila caspase dronc. Cell Death Differ. 10: 1348-1356.

Cakouros D, Daish TJ, Mills K, Kumar S (2004) An arginine-histone methyl transferase, CARMER, coordinates ecdysone-mediated apoptosis in Drosophila cells. J. Biol. Chem. 279:18467-18471.

Cakouros D, Daish TJ, Kumar S (2004) Ecdysone receptor directly binds the promoter of the Drosophila caspase dronc regulating its expression in specific tissues. J. Cell Biol. 165: 631- 640.

Ekert PG, Read SH, Silke J, Marsden V, Kaufmann H, Hawkins CJ, Gerl R, Kumar S, Vaux DL (2004) Apaf-1, caspase-2 and caspase-9 accelerate apoptosis, but do not determine whether factor-deprived or drug-treated cells die. J. Cell Biol. 165: 835-842.

Murdaca J, Treins C, Monthouël-Kartmann MN, Pontier-Bres R, Kumar S, Van Obberghen E, Giorgetti-Peraldi S (2004) Grb10 prevents Nedd4-mediated VEGF receptor-2 degradation. J. Biol. Chem. 279: 26754-26761.

Fotia AB, Ekberg J, Cook DI, Adams DJ, Poronnik P, Kumar S (2004) Regulation of neuronal voltage-gated sodium channels by the ubiquitin-protein ligases Nedd4 and Nedd4-2. J. Biol. Chem. 279: 28930-28935.

Kumar S, Cakouros D (2004) Transcriptional control of the core cell death machinery. Trends Biochem. Sci. 29: 193-199.

Kumar S (2004) Migrate, differentiate, proliferate or die: Pleiotropic functions of an apical “apoptotic caspase”. Science STKE 2004: pe49.

Shearwin-Whyatt LM, Brown DL, Wylie FG, Stow JL, Kumar S (2004) N4WBP5A (Ndfip2), a Nedd4-interacting protein, localizes to multivesicular bodies and the Golgi, and has a potential role in protein trafficking. J. Cell Sci. 117: 3679-3689.

Dinudom A, Fotia A, Lefkowitz RJ, Young JA, Kumar S, Cook DI (2004) The kinase Grk2 regulates the Nedd4/Nedd4-2 dependent control of epithelial Na+ channels. Proc. Natl. Acad. Sci. USA. 101: 11886-11890.

Baliga BC, Read SH, Kumar S (2004) The biochemical mechanism of caspase-2 activation. Cell Death Differ. 11: 1234-1241.

Dorstyn L, Mills K, Lazebnik Y, Kumar S (2004) The two cytochrome c species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells. J. Cell Biol. 167: 405-410.

Chew SK, Akdemir F, Chen P, Lu, W-J, Mills K, Daish, T, Kumar S, Rodriguez A, Abrams JM (2004) The apical caspase, dronc, governs programmed and unprogrammed cell death in Drosophila. Developmental Cell 7: 897-907.

Daish TJ, Mills K, Kumar S (2004) Drosophila caspase DRONC is required for specific developmental cell death pathways and stress-induced apoptosis. Developmental Cell 7: 909-915.

Hryciw DH, Ekberg J, Lee A, Lensink IL, Kumar S, Guggino WB, Cook DI, Carol A. Pollock CA, Poronnik P (2004) Nedd4-2 functionally interacts with ClC-5: involvement in constitutive albumin endocytosis in proximal tubule cells. J. Biol. Chem. 279: 54996-55007.

Dorstyn L, Kumar S (2004) Programmed cell death in Drosophila melanogaster. In: When Cells Die II- A comprehensive evaluation of apoptosis and programmed cell death (R. A. Lockshin and Z. Zakeri eds.), John Wiley & Sons, Inc., New York, pp 79-97.

Kumar S (2004) Measurement of caspase activity in cells undergoing apoptosis. In: Apoptosis Methods and Protocols (H.J.M. Brady ed.), Methods in Molecular Biology 282:19-30.

2003

Baliga C, Kumar S (2003) The Apaf-1/cytochrome c apoptosome: an essential initiator of caspase activation or just a sideshow? Cell Death Differ. 10: 15-17.

Fotia A, Dinudom A, Shearwin KE, Koch J-P, Korbmacher C, Cook DI, Kumar S (2003) The role of individual Nedd4-2 (KIAA0439) WW domains in binding and regulating epithelial sodium channels. FASEB J. 17: 70-72.

Baliga BC, Colussi PA, Read SH, Dias MM, Jans DA, Kumar S (2003) Role of prodomain in importin-mediated nuclear localization and activation of caspase-2. J. Biol. Chem. 278: 4899-4905.

Kim TW, Hung C-F, Ling M, Juang J, He L, Hardwick JM, Kumar S, Wu T-C (2003) Enhancing DNA vaccine potency by coadministration of DNA encoding antiapoptotic proteins. J. Clin. Invest. 112: 110-117.

Quinn L, Coomb M, Mills K, Daish T, Colussi P, Kumar S, Richardson H (2003) Buffy, a Drosophila Bcl-2 related protein, has anti-apoptotic and cell cycle inhibitory function. EMBO J. 22: 3568-3579.

Daish T, Cakouros D, Kumar S (2003) Distinct promoter regions regulate spatial and temporal expression of the Drosophila caspase dronc. Cell Death Differ. 10: 1348-1356.

2002

Shcherbik N, Kumar S, Haines DS (2002) Substrate proteolysis is inhibited by dominant-negative Nedd4 and Rsp5 mutants harboring alterations in WW domain 1. J. Cell Sci. 115: 1041-1048.

Harvey KF, Shearwin-Whyatt LM, Fotia A, Parton RG, Kumar S (2002) N4WBP5, a potential target for ubiquitination by the Nedd4 family of proteins, is a novel Golgi-associated protein. J. Biol. Chem. 277: 9307-9317.

Dorstyn L, Read S, Cakouros D, Huh JR, Hay BA, Kumar S (2002) The role of cytochrome c in caspase activation in Drosophila cells. J. Cell Biol. 156: 1089-1098.

O'Reilly LA, Ekert P, Harvey N, Marsden V, Cullen L, Vaux DL, Hacker G, Magnusson C, Pakusch M, Cecconi F, Strasser A, Huang DCS, Kumar S (2002) Caspase-2 is not required for thymocyte or neuronal apoptosis even though cleavage of caspase-2 is mediated by Apaf-1 and caspase-9. Cell Death Diff. 9: 832-841.

Cakouros D, Daish T, Martin D, Baehrecke EH, Kumar S (2002) Ecdysone-induced expression of the caspase Dronc during hormone dependent programmed cell death in Drosophila is regulated by Broad-Complex. J. Cell Biol. 157: 985-995.

Baliga BC, Kumar S (2002) Role of Bcl-2 family of proteins in malignancy. Hematol. Oncology. 20: 63-74.

Richardson H, Kumar S (2002) Death to flies: Drosophila as a model system to study programmed cell death. J. Immunol. Meth. 265: 21-38.

Konstas A-A, Shearwin-Whyatt LM, Fotia A, Degger B, Riccardi D, Cook DI, Korbmacher C, Kumar S (2002) Regulation of the epithelial sodium channel by N4WBP5A, a novel Nedd4/Nedd4-2-interacting protein. J. Biol. Chem. 277: 29406-29416.

Read SH, Baliga BB, Ekert P, Vaux DL, Kumar S (2002) A novel Apaf-1-independent putative caspase-2 activation complex. J. Cell Biol. 159: 739-745.

Kumar S, Vaux DL (2002) A Cinderella caspase takes center stage. Science 297: 1290-1291.

Selected earlier publication

Dinudom A, Harvey KF, Komwatana P, Jolliffe CN, Young JA, *Cook DI, *Kumar S (2001) The roles of the carboxyl termini of a-, b- and g-subunits of epithelial Na+ channels (ENaC) in regulating ENaC and in mediating its inhibition by cytosolic Na+. J. Biol. Chem. 276: 13744-13749. (*equal senior authors)

Harvey KF, Dinudom A, Cook DI, Kumar S (2001) The Nedd4-like protein KIAA0439 is a potential regulator of the epithelial sodium channel. J. Biol. Chem. 276: 8597-8601.

Doumanis J, Quinn L, Richardson H, Kumar S (2001) STRICA, a novel Drosophila caspase with an unusual serine/threonine-rich prodomain, interacts with DIAP1 and DIAP2. Cell Death Diff. 8: 387-394.

Harvey NL, Daish T, Mills K, Dorstyn L, Quinn LM, Read SH, Richardson H, Kumar S (2001) Characterization of the Drosophila caspase, DAMM. J. Biol. Chem. 276: 25342-25350.

Shearwin-Whyatt LM, Harvey NL, Kumar S (2000) Subcellular localization and CARD-dependent oligomerization of the death adaptor RAIDD. Cell Death Diff. 7: 155-165.

Kumar S, Doumanis J (2000) The fly caspases. Cell Death Differ. 7: 1039-1044.

Colussi PA, Quinn LM, Huang DCS, Coombe M, Read SH, Richardson H, Kumar S (2000) Debcl, a pro-apoptotic Bcl-2 homologue, is a component of the Drosophila cell death machinery. J. Cell Biol. 148: 703-714.

Jolliffe CN, Harvey KF, Haines BP, Parasivam G, Kumar S (2000) Identification of multiple proteins expressed in murine embryos as binding partners for the WW domains of the ubiquitin-protein ligase Nedd4. Biochem. J. 351: 557-565.

Quinn LM, Dorstyn L, Mills K, Colussi PA, Chen P, Coombe M, Abrams J, *Richardson H, *Kumar S (2000) An essential role for the caspase Dronc in developmentally programmed cell death in Drosophila. J. Biol Chem.275: 40416-40424. (*equal senior authors)

Kumar S, Colussi PA (1999) Prodomains-adaptors-oligomerisation: the pursuit of caspase activation in apoptosis. Trends Biochem. Sci. 24: 1-4.

Harvey KF, Dinudom A, Komwatana P, Jolliffe CN, Day ML, Parasivam G, Cook DI, Kumar S (1999) All three WW domains of murine Nedd4 are involved in the regulation of epithelial sodium channels by intracellular Na+. J. Biol. Chem. 274: 12525-12530.

Dorstyn L, Colussi PA, Quinn LM, Richardson H, Kumar S (1999) DRONC, an ecdysone-inducible Drosophila caspase. Proc. Natl. Acad. Sci. USA 96: 4307-4312.

Ishibashi H, Dinudom A, Harvey KF, Kumar S, Young JA, Cook DI (1999) Na+-H+ exchange in salivary secretary cells is controlled by an intracellular Na+ receptor. Proc. Natl. Acad. Sci. USA 96: 9949-9953.

Morrione A, Plant P, Valentinis B, Staub O, Kumar S, Rotin D and Baserga R (1999) mGrb10 interacts with Nedd4. J. Biol. Chem. 274: 24094-24099.

Dorstyn L, Read SH, Quinn LM, Richardson H, Kumar S (1999) DECAY, a novel Drosophila caspase related to mammalian caspase-3 and caspase-7. J. Biol. Chem. 274: 30778-30783.

Harvey KF, Kumar S (1999) Nedd4-like proteins: an emerging family of ubiquitin-protein ligases implicated in diverse cellular functions. Trends Cell Biol. 9: 166-169.

Kumar S (1999) Mechanisms of caspase activation in cell death. Cell Death Diff. 6: 1060-1066.

Dinudom A, Harvey KF, Komwatana P, Young JA, Kumar S, Cook DI (1998) Nedd4 mediates control of an epithelial Na+ channel in salivary duct cells by cytosolic Na+. Proc. Natl. Acad. Sci. USA 95: 7169-7173.

Butt AJ, Harvey NL, Parasivam G, Kumar S (1998) Dimerization and autoprocessing of the Nedd2 (caspase-2) precursor requires both the prodomain and the carboxyl terminal regions. J. Biol. Chem. 273: 6763-6768.

Harvey KF, Harvey NL, Michael JM, Parasivam G, Waterhouse N, Alnemri ES, Watters D, Kumar S (1998) Caspase-mediated cleavage of the ubiquitin-protein ligase Nedd4 during apoptosis. J. Biol. Chem. 273: 13524-13530.

Bird CH, Sutton VR, Sun J, Hirst CE, Novak A, Kumar S, Trapani JA, Bird PI (1998) Selective regulation of apoptosis: the cytotoxic lymphocyte serpin PI-9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway. Mol. Cell. Biol. 18: 6387-6398.

Colussi PA, Harvey NL, Kumar S (1998) Prodomain-dependent nuclear localization of the caspase-2 (Nedd2) precursor: A novel function for a caspase prodomain. J. Biol. Chem. 273: 24535-24542.

Waterhouse NJ, Finucane D, Green DR, Elce JS, Kumar S, Alnemri E, Litwack G, Lavin MF, Watters D (1998) Calpain activation is upstream of caspases in irradiation-induced apoptosis. Cell Death Diff. 5: 1051-1061.

Colussi PA, Harvey NL, Shearwin-Whyatt LM, Kumar S (1998) Conversion of procaspse-3 to an autoactivating caspase by fusion to the caspase-2 prodomain. J. Biol. Chem. 273: 26566-26570.

Kumar S, Kinoshita M, Dorstyn L, Noda M (1997) Origin, expression and possible functions of the two alternatively spliced forms of the mouse Nedd2 mRNA. Cell Death Diff. 4: 378-387.

Kumar S, Harvey KF, Kinoshita M, Copeland NG, Noda M, Jenkins NA (1997) cDNA cloning, expression analysis and mapping of the mouse Nedd4 gene. Genomics 40: 435-443.

Kinoshita M, Kumar S, Mizoguchi A, Ide C, Kinoshita A, Haraguchi T, Hiraoka Y, Noda M (1997) Nedd5, a mammalian septin, is a novel cytoskeletal component interacting with actin-based structures. Genes Dev. 11: 1535-1547.

Harvey NL, Butt A, Kumar S (1997) Functional activation of Nedd2/ICH-1 (caspase-2) is an early process in apoptosis. J. Biol. Chem. 272: 13134-13139.

Kumar S, Lavin MF (1996) The ICE family of cysteine proteases as effectors of cell death. Cell Death Diff. 3: 255-267.

Song Q, Lees-Miller SP, Kumar S, Zhang N, Chan DW, Smith GCM, Jackson SP, Alnemri ES, Litwack G, Khanna KK, Lavin MF (1996) DNA-dependent protein kinase catalytic subunit: A target for an ICE-like protease in apoptosis. EMBO J.  15: 3238-3246.

Song Q, Burrows S, Lees-Miller S, Smith G, Jackson S, Kumar S, Trapani JA, Alnemri E, Litwack G, Lu H, Moss D, Lavin MF (1996) ICE-like protease cleaves DNA-dependent protein kinase in cytotoxic T-cell killing. J. Exp. Med. 184: 619-626.

Waterhouse N, Kumar S, Song Q, Strike P, Sparrow L, Dreyfuss G, Alnemri ES, Litwack G, Lavin M, Watters D (1996) Heteronuclear ribonucleoproteins C1 and C2, components of the spliceosome, are specific targets of interleukin-1beta-converting enzyme-like proteases in apoptosis. J. Biol. Chem. 271: 29335-29341.

Kumar S (1995) ICE-like proteases in apoptosis. Trends Biochem. Sci. 20: 198-202.

Kumar S, Matsuzaki T, Yoshida Y, Noda M (1994) Molecular cloning and biological activity of a novel developmentally regulated gene encoding a protein with transducin beta-like structure. J. Biol. Chem. 269: 11318-11326.

Kumar S, Kinoshita M, Noda M, Copeland NG, Jenkins NA (1994) Induction of apoptosis by mouse Nedd2 gene, which encodes a protein similar to the product of the Caenorhabditis elegans cell death gene ced-3 and the mammalian IL-1beta-converting enzyme. Genes Dev. 8: 1613-1626.

Kumar S, Yoshida Y, Noda M (1993) Cloning of a cDNA which encodes a novel ubiquitin-like protein. Biochem. Biophys. Res. Commun. 195: 393-399.

Kumar S, Tomooka Y, Noda M (1992) Identification of a set of genes with developmentally down-regulated expression in the mouse brain. Biochem. Biophys. Res. Commun. 185: 1155-1161.

See a PubMed listing of Professor Sharad Kumars's publications

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Student Projects

The Molecular Regulation Laboratory provides an outstanding environment for Honours and Postgraduate studies. For individual projects, prospective students should contact the laboratory staff. Scholarships are available through various sources including those listed below:


1. The role of caspase-2 in specific pathways of apoptosis and tumorigenesis.

(Contact: Dr Loretta Dorstyn, mailto:loretta.dorstyn@imvs.sa.gov.au

Our ongoing work has made a landmark discovery that the lack of caspase-2 enhances the ability of cells to transform readily and that caspase-2 deficiency increases the potential of tumourigenesis in vivo. Using the mouse E�-Myc lymphoma model we found that the loss of even a single allele of caspase-2 resulted in accelerated tumourigenesis, and this was further enhanced in caspase-2-/- mice. The caspase-2-/- cells show increased growth rates, a defective apoptotic response to cell cycle checkpoint regulation and show abnormal cycling following �-irradiation. These data show that loss of caspase-2 results in an increased ability of cells to acquire a transformed phenotype and become malignant, indicating that caspase-2 is a tumour suppressor protein. This project involves testing whether the tumour suppressor function of caspase-2 is limited to only Myc-induced lymphomagenesis, or is more general. Cellular and molecular studies will dissect out the mechanism by which caspase-2 acts as a tumour suppressor and identify target(s) of caspase-2 that mediate its tumour suppressor function.


2. Cell death regulation during animal development.

(Contact: Dr Donna Denton, donna.denton@imvs.sa.gov.au or Dr Saman Ibrahimi, mailto:donna.denton@imvs.sa.gov.au)

We have been utilising Drosophila as an in vivo model to dissect out the mechanisms of developmentally programmed cell death (PCD). Our ongoing studies have led to several seminal findings, including the discovery of the key canonical pathway of PCD involving the caspase Dronc, and the adaptor Ark. We have also discovered a novel potential regulator of caspase activation, CG4230, from a Dronc-interaction screen. This project involves the characterisation of the role of CG4230 in caspase activation and cell death, and identify other potential regulators of caspase activation


3. Role of autophagy in PCD.

(Contact: Dr Donna Denton, donna.denton@imvs.sa.gov.au)

In recent studies we discovered that the Dronc/Ark pathway, while essential for most PCD, is largely dispensable for developmental PCD in specific tissues. This is most obvious in the larval midgut (MG), which undergoes PCD during metamorphosis, and this process is unaffected in dronc and ark mutants. In preliminary studies we have found that the inhibition of autophagy leads to a delay in MG removal indicating a potential role for autophagy in MG PCD. Given that the role of autophagy in cell death is a matter of extensive debate, our discovery that MG PCD can be delayed by genetically blocking autophagy provides a unique model for delineating this controversy. We hypothesise that during development PCD utilises caspase-dependent (most tissues), caspase- and autophagy-dependent (e.g. larval salivary glands) and caspase-independent but autophagy-dependent (e.g. midgut) mechanisms. In this project we will delineate the mechanism of midgut cell death by exploring the contribution of caspases and autophagy. The project also aims to define the role of growth signalling in midgut cell death.


4.The role of Nedd4 and Nedd4-2 in hypertension and the regulation of sodium channels.

(Contact: Dr Natasha Boase, natasha.boase@imvs.sa.gov.au)

Aberrations in the ubiquitin system underpin the pathogenesis of many diseases including cancer, neurodegenerative disorders and channelopathies. The Nedd4 and related ubiquitin ligases (E3s) are required for the ubiquitination of numerous cellular targets involved in processes such as transcription, stability and trafficking of plasma membrane proteins, and the degradation of misfolded proteins. We have shown that Nedd4-2 E3 ubiquitinates the epithelial sodium channel (ENaC). ENaC is required for sodium absorption across a range of epithelial tissues such as the lungs, colon and kidney and is an important regulator of blood sodium concentration and blood pressure. Ubiquitination of ENaC by Nedd4 and Nedd4-2 leads to its internalisation and degradation. Our current focus is to characterise the mechanisms of regulation of ENaC by Nedd4-2 in vivo by using Nedd4-2 gene knockout mice.


5. Regulation of animal growth by Nedd4.

(Contact: Dr Natasha Boase, natasha.boase@imvs.sa.gov.au)

In a collaborative study with Prof Yang (University of Iowa) we have recently found that the loss of Nedd4 in mice results in reduced IGF-1 and insulin signalling, reduced growth and neonatal lethality. Nedd4-deficient cells show reduced mitogenic activity. This appears to be due to increased levels of the adaptor protein Grb10 resulting in IGF-1R mislocalization and inhibition of IGF-1 and insulin signalling. We are now studying the mechanism of Grb10 regulation by Nedd4. There is evidence to suggest that Nedd4 has additional cellular targets. Thus, we are analysing additional phenotypes that may be associated with the knockout of Nedd4. This project aims to decipher the mechanisms by which Nedd4 controls animal growth and development.


6. The regulation of iron homeostasis by the Nedd4 binding proteins, Ndfip1 and Ndfip2.

(Contact: Dr Natalie Foot, natalie.foot@imvs.sa.gov.au or Dr Hazel Dalton, hazel.dalton@imvs.sa.gov.au)

Iron homeostasis is a highly regulated process which, if perturbed, leads to a number of disease states such as haemochromatosis or anaemia. Our recent work shows that Ndfips regulate the divalent metal ion transporter DMT1, the primary non-heme iron transporter in mammals. DMT1 interacts with both Ndfip1 and Ndfip2, and this promotes DMT1 ubiquitination and degradation by the Nedd4-family ubiquitin ligase, WWP2. Furthermore the Ndfip1 knockout mice show increased hepatic iron deposition, indicating an essential function of Ndfip1 in iron homeostasis. Given that misregulation of DMT1 is implicated in a number of human diseases, Ndfips and WWP2 could be potential targets for therapeutic intervention to control iron uptake and/or metabolism. Our current focus is to further characterise WWP2 and Ndfip knockout mice to provide additional understanding of this novel mechanism of regulating iron transport, and to further delineate the regulation of DMT1 by Ndfips and WWP2.


Scholarships are available through various sources including those listed below:

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Funding