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Dr Stuart M Pitson

Address	Rm 3-64-S Division of Human Immunology Institute of Medical and Veterinary Science Frome Road, Adelaide SA 5000, Australia
Phone	+61 8 8222 3472
Fax	+61 8 8232 4092
Email	Stuart.Pitson@imvs.sa.gov.au
	Research Interests

Affiliations:

Member, Hanson Institute.

Affiliate Senior Lecturer, School of Molecular and Biomedical Science, University of Adelaide.

Qualifications: B.App.Sc., Ph.D. (LaTrobe)

Honours:

Experience:

Scientific Involvement:

Editorial:

Lab Members

Research Interests

The Molecular Signalling Laboratory examines sphingolipid-mediated cell signalling pathways, and how they contribute to cancer, inflammatory diseases, hypertension and other medical conditions. In particular, the enzyme sphingosine kinase is the primary focus of our work. This enzyme catalyses the formation of the phospholipid signalling molecule, sphingosine 1-phosphate.

Sphingosine 1-phosphate regulates a diverse range of cellular processes through its roles as both a ligand for a family of sphingosine 1-phosphate-specific cell surface receptors, as well as an intracellular second messenger. Of greatest interest to our laboratory are findings that elevated cellular sphingosine kinase (and sphingosine 1-phosphate) prevents programmed cell death (apoptosis), enhances cell proliferation, and leads to neoplastic cell transformation. This indicates an oncogenic role for sphingosine kinase, which is further supported by recent data showing elevated sphingosine kinase in a variety of human cancer cells and inhibition of tumor growth in vivo by sphingosine kinase inhibitors.

In addition to this role in tumorigenesis, sphingosine kinase and sphingosine 1-phosphate appear central players in many other cellular processes, including; vascular endothelial cell activation, a hallmark of inflammatory diseases; enhancing blood vessel construction, and; enhancing constriction of airway smooth muscle cells. Thus, sphingosine kinase is also a potential target for therapeutic intervention in inflammation and atherosclerosis, hypertension and asthma.

Current work in this laboratory is concentrated on understanding the biochemistry of sphingosine kinase, identifying the mechanisms regulating the activity and localisation of this enzyme, and on the (patho-)physiological functions of signal transduction pathways it controls. Understanding these factors may allow for the development of novel anti-sphingosine kinase therapeutics. Much of our work to date on sphingosine kinase has focused on the post-translational regulation of this enzyme. Sphingosine kinase is activated in cells in response to certain growth factors and other agonists. We have shown that activation of sphingosine kinase 1 occurs through Ser225 phosphorylation by ERK1/2 which not only enhances its catalyic activity, but also results in its translocation to the plasma membrane. We have made a major breakthrough by demonstrating that this phosphorylation, and especially the subsequent translocation, mediates the pro-proliferative, pro-survival and oncogenic effects of sphingosine kinase 1. However, the mechanism(s) regulating the phosphorylation status of SK1 and its translocation are not known, and are one of the primary foci of our current studies.

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Some recent and current projects carried out in this laboratory

1. Identification of the nucleotide-binding site of sphingosine kinase

Despite the importance of sphingosine kinase 1, very little is known regarding its structure or mechanism of catalysis. Moreover, sphingosine kinase 1 does not contain recognisable catalytic or substrate binding sites, based on sequence motifs found in other kinases. We have elucidated the nucleotide-binding site of human sphingosine kinase through a combination of site-directed mutagenesis and affinity labelling with an ATP analogue . While sharing some sequence and likely weak structural similarity with the highly conserved nucleotide-binding site of many protein kinases, the nucleotide-binding site of sphingosine kinase is unique. This finding raises the possibility of generating specific inhibitors of sphingosine kinase activity through targeting the nucleotide-binding site.

• Pitson SM, Moretti PAB, Zebol JR, Zareie R, Derian CK, Darrow AL, Qi J, D'Andrea RJ, Bagley CJ, Vadas MA and Wattenberg BW (2002) The nucleotide-binding site of human sphingosine kinase 1. J Biol Chem 277, 49545–49553.
  

2. Generation of a dominant-negative sphingosine kinase

We have developed and characterised a catalytically inactive mutant of sphingosine kinase 1 that, when overexpressed in cells, blocks agonist-induced activation of endogenous sphingosine kinase activity. This finding has enabled more precise examination of the cellular pathways effected by sphingosine 1-phosphate and sphingosine kinase activation since the previously available chemical inhibitors had only poor specificity.

• Pitson SM, Moretti PAB, Zebol JR, Xia P, Gamble JR, Vadas MA, D'Andrea RJ and Wattenberg BW (2000) Expression of a catalytically inactive sphingosine kinase mutant blocks agonist-induced sphingosine kinase activation: a dominant-negative sphingosine kinase. J Biol Chem 275, 33945–33950.

3. The catalytic and functional activation of sphingosine kinase 1 by phosphorylation

We have identified that phosphorylation of sphingosine kinase 1 at serine-225 by a member of the extracellular signal regulated protein kinase (ERK) family directly results in its activation. Strikingly, the ability of overexpressed sphingosine kinase 1 to support enhanced proliferation, survival, and neoplastic cell transformation is blocked by mutation of the phosphorylation site. This is despite this non-phosphorylatable mutant retaining full basal catalytic activity. More recently we have established that this single phosphorylation of sphingosine kinase 1 not only directly increases its catalytic activity but also results in its translocation from the cytosol to the plasma membrane. Furthermore, we have shown that this phosphorylation-induced change in localisation of sphingosine kinase 1 is critical in driving oncogenic signalling by this enzyme.

• Pitson SM, Moretti PAB, Zebol JR, Lynn HE, Xia P, Vadas MA and Wattenberg BW (2003) Activation of sphingosine kinase 1 by ERK1/2-mediated phosphorylation. EMBO J 22, 5491–5500.
• Pitson SM, Xia P, Leclercq TM, Moretti PAB, Zebol JR, Lynn HE, Wattenberg BW and Vadas MA (2005) Phosphorylation-dependent translocation of sphingosine kinase to the plasma membrane drives its oncogenic signalling. J Exp Med 201, 49–54.

4. Identification of the calmodulin-binding site of sphingosine kinase

We have examined the known Ca2+-dependent interaction of sphingosine kinase 1 with calmodulin (CaM), and using a combination of limited proteolysis, peptide interaction analysis and site-directed mutagenesis, have identified the unique CaM-binding site of this enzyme. We have also shown for the first time that sphingosine kinase 2 also binds CaM, and does so via a region that is conserved with sphingosine kinase 1. Furthermore, using the CaM-binding-deficient version of sphingosine kinase 1 we have begun to elucidate the role of CaM in sphingosine kinase 1 regulation by demonstrating that disruption of the CaM-binding site ablates agonist-induced translocation of sphingosine kinase 1 from the cytoplasm to the plasma membrane. This indicates that CaM, or a CaM-like protein, is essential in the translocation of sphingosine kinase 1 which appears critical for the signalling functions of this enzyme.

• Sutherland CM, Moretti PAB, Hewitt NM, Bagley CJ, Vadas MA and Pitson SM (2006) The calmodulin binding site of sphingosine kinase and its role in agonist-dependent translocation of sphingosine kinase 1 to the plasma membrane. J Biol Chem 281, 11693–11701.

5. Identification of a sphingosine kinase-interacting proteins that may play a role in sphingosine kinase 1 regulation

We have identified several proteins that interact with sphingosine kinase 1 through the use of a yeast two-hybrid screen. We are currently examining some of these sphingosine kinase 1-interacting proteins to establish their possible roles in the regulation of sphingosine kinase 1 activity and function.

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Selected Recent Publications

Pitson SM, D'Andrea RJ, Vandeleur L, Moretti PAB, Xia P, Gamble JR, Vadas MA and Wattenberg BW (2000) Human sphingosine kinase: purification, molecular cloning and characterisation of the native and recombinant enzymes. Biochem J 350, 429–441.

Pitson SM, Moretti PAB, Zebol JR, Xia P, Gamble JR, Vadas MA, D'Andrea RJ and Wattenberg BW (2000) Expression of a catalytically inactive sphingosine kinase mutant blocks agonist-induced sphingosine kinase activation: a dominant-negative sphingosine kinase. J Biol Chem 275, 33945–33950.

Xia P, Gamble JR, Wang L, Pitson SM, Moretti PAB, D'Andrea RJ, Wattenberg BW and Vadas MA (2000) An oncogenic role of sphingosine kinase. Curr Biol 10, 1527–1530.

Pitson SM, Moretti PAB, Zebol JR, Zareie R, Derian CK, Darrow AL, Qi J, D'Andrea RJ, Bagley CJ, Vadas MA and Wattenberg BW (2002) The nucleotide-binding site of human sphingosine kinase 1. J Biol Chem 277, 49545–49553.

Pitson SM, Moretti PAB, Zebol JR, Lynn HE, Xia P, Vadas MA and Wattenberg BW (2003) Activation of sphingosine kinase 1 by ERK1/2-mediated phosphorylation. EMBO J 22, 5491–5500.

Pitson SM, Xia P, Leclercq TM, Moretti PAB, Zebol JR, Lynn HE, Wattenberg BW and Vadas MA (2005) Phosphorylation-dependent translocation of sphingosine kinase to the plasma membrane drives its oncogenic signalling. J Exp Med 201, 49–54.

Ma Y, Pitson S, Hercus T, Murphy J, Lopez A and Woodcock J (2005) Sphingosine activates PKA type II by a novel cAMP-independent mechanism. J Biol Chem 280, 26011–26017.

Pébay A, Wong RCB, Pitson SM, Wolvetang EJ, Peh GSL, Filipczyk A, Koh KLL, Tellis I, Nguyen LTV and Pera MF (2005) Essential roles of sphingosine-1-phosphate and platelet-derived growth factor in the maintenance of human embryonic stem cells. Stem Cells 23, 1541–1548.

Sutherland CM, Moretti PAB, Hewitt NM, Bagley CJ, Vadas MA and Pitson SM (2006) The calmodulin binding site of sphingosine kinase and its role in agonist-dependent translocation of sphingosine kinase 1 to the plasma membrane. J Biol Chem 281, 11693–11701.

Wattenberg BW, Pitson SM and Raben DM (2006) The sphingosine and diacylglycerol kinase superfamily of signaling kinases: localization as a key to signaling function. J Lipid Res 47, 1128–1139.

Leclercq TM and Pitson SM (2006) Cellular signalling by sphingosine kinase and sphingosine 1-phosphate. iubmb Life 58, 467–472.

Soldi R, Mandinova A, Venkataraman K, Hla T, Vadas MA, Pitson SM, Duarte M, Graziani I, Kolev V, Kacer D, Kirov A, Maciag T and Prudovsky I (2007) Sphingosine kinase 1 is a critical component of the copper-dependent FGF1 export pathway. Exp Cell Res 313, 3308–3318.

Pébay A, Bonder CS and Pitson SM (2007) Stem cell regulation by lysophospholipids. Prostaglandins & Other Lipid Mediators 84, 83–97.

Leclercq TM, Moretti PAB, Vadas MA and Pitson SM (2008) Eukaryotic elongation factor 1A interacts with sphingosine kinase and directly enhances its catalytic activity. J Biol Chem 283, 9606–9614.

Pham DH, Moretti PAB, Goodall GJ and Pitson SM (2008) Attenuation of leakiness in doxycycline-inducible expression by incorporation of 3’ AU-rich mRNA destabilizing elements. Biotechniques, in press.

See a PubMed listing of Dr Stuart Pitson's publications

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Funding

	SDP Technologies

Available Student Projects

1. The molecular mechanisms of sphingosine kinase regulation
Sphingosine kinase becomes rapidly and transiently activated in cells in response to growth factors and other regulatory agonists. This activation is critical in the signalling functions of this enzyme, and its disregulation can lead to tumor formation. Thus, knowing how this activation occurs is important for understanding the function of this enzyme. We have recently made a major advance in this area by establishing that phosphorylation of sphingosine kinase at serine-225 by a member of the extracellular signal regulated protein kinase (ERK) family directly results in its activation. Much is still not known, however, regarding how this phosphorylation is regulated, and whether other alternative regulatory mechanisms also control the activity and cellular location of this protein. Indeed, we have recently identified several proteins that interact with sphingosine kinase through the use of a yeast two-hybrid screen. We are currently examining some of these proteins to establish their possible roles in the regulation of sphingosine kinase activity and function.

2. The cell signalling pathways controlled by sphingosine kinase
Sphingosine kinase is involved in the development of a number of disease states, including cancer, inflammation and atherosclerosis, asthma and hypertension. The exact mechanisms whereby sphingosine kinase exerts these effects, however, are relatively poorly understood. Thus, we are undertaking studies to better understand which cell signalling pathways are controlled by sphingosine kinase and its product, sphingosine 1-phosphate, and how this regulation is achieved. This involves both (i) biochemical studies to directly identify the specific intracellular targets regulated by sphingosine kinase and sphingosine 1-phosphate (ie. sphingosine 1-phosphate binding proteins), as well as (ii) microarray and phosphoprotein array studies to identify the broader pathways regulated by sphingosine kinase and its activation. For these latter experiments we have developed a large number of important molecular tools to definitively disect the signalling pathways regulated by sphingosine kinase, its activation, and its agonist-induced translocation to the plasma membrane.

Join the Australian Society for Biochemistry and Molecular Biology

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