PI3-kinase catalyses the addition of a phosphate group on the 3' position of phosphatidylinositol (PI) and the phosphoinositides PI(4)P and PI(4,5)P2. Elevated levels of the products of this enzyme, especially PI(3,4,5)P3, have been observed immediately following growth factor receptor activation and also in cells transformed by a variety of oncogenes. The role of these products in transducing an anti-apoptotic and pro-mitogenic signal is realized through their interaction with specific serine/threonine kinases (called PDK1 and Akt/PKB), which phosphorylate and modulate the activity of a variety of proteins involved in growth and survival control.

We follow various approaches to elucidate these pathways further. For example, a proteomic approach is used to identify proteins that are differentially expressed, degraded or translocated within cells upon their activation by various growth factors. We employ the techniques of sub-cellular fractionation, 2-dimensional gel electrophoresis and mass spectroscopy to record changes in the patterns of protein expression and investigate the effects of specific inhibitors of PI 3-kinase. Identified proteins are further analyzed to understand the role they play in the growth factor response. We have extended this methodology using cells from transgenic and knockout mice in which specific components of the PI3K pathway, such as Akt, have either been over-expressed or inactivated. Another approach involves the characterization of large multiprotein complexes formed by components of this pathway using the Tandem Affinity Purification method and protein identification by mass spectroscopy

The MAPK cascade

The family of the mitogen-activated protein kinases (MAPK) includes the c-Jun N-terminal protein kinases (JNK) that are activated by mitogenic stimuli, pro-inflammatory cytokines and a variety of environmental stresses. These kinases are key regulators of the activities of many transcription factors, which include c-Jun, ATF2 and Elk1, controlling cell growth and apoptosis. An improved understanding of the JNK-mediated signaling may provide novel strategies in prevention and treatment of cancer and other diseases. As well as being regulated positively by phosphorylation, regulation of this kinase at the level of dephosphorylation is likely to be very important. Inhibition of phosphatases that dephosphorylate JNK could be part of the mechanism of increasing JNK activity. It has been shown that exposure of cells to protein damaging stresses including arsenite, oxidative stress and heat shock, strongly reduces the rate of JNK dephosphorylation. Regulation of the JNK pathway by arsenite may be relevant in carcinogenesis since chronic arsenic exposure is associated with increased risk of human cancer of the skin, bladder, kidney, lung and colon. A dual-specificity phosphatase (M3/6) has been identified, showing specificity towards JNK. The activity of this phosphatase, is inhibited by arsenite, thus leading to an increase in JNK phosphorylation and activity. We aim to determine the exact mechanism of arsenite regulation of the M3/6 phosphatase activity and to identify additional arsenite-sensitive JNK specific phosphatases. This should lead to a better understanding of the role of arsenite in tumor promotion. To achieve this aim we are investigating:

The regulation of the enzymatic activity of M3/6 by phosphorylation upon stimulation with arsenite and mapping of the relevant phosphorylation sites by mass spectrometry.
Analysis of the interaction of M3/6 with JNK through two identified candidate regions within the M3/6 sequence.
Use of JNK specific phosphatases to study JNK activation and transformation, through conditional expression of the phosphatases in oncogene-transformed cells.
Identification of M3/6 - interacting proteins by TAP and mass spectroscopy.
The in vivo role of JNK-specific dual-specificity phosphatases, using Drosophila and knock-out mice as model systems.