The phosphoinositide 3-kinase (PI3K) pathway has been established as a core signaling pathway that governs cell growth, cell proliferation, survival and differentiation in all cell types. Not surprisingly, dysregulation of this pathway in humans, as exemplified by loss of function of PTEN (Phosphatase and TENsin homolog), a prominent phosphoinositide 3-phosphatase tumor suppressor that directly inhibits PI3K signaling, results in diverse pathological conditions. Patients carrying inactivating mutations of PTEN have a prevalence to develop tumors that can coincide with neurological defects such as mental retardation, ataxia and seizure. On the other hand, inhibition of PTEN activity is currently seen as a persuasive target for increasing regenerative capacities of neurons affected in degenerative conditions or following injury to the nervous system. We have identified and initially validated a number of novel neuronal interacting proteins that associate with PTEN and we are currently delevoping methods to probe these novel protein-protein interactions, using homogeneous purified populations of embryonic stem (ES) cell-derived neurons. We are using an inducible expression system to ectopically express candidate genes and create transgenic ES cells that can then be differentiated into motor neurons in vitro. Isolation of purified populations is achieved by magnetic-activated cell sorting, using a CD14 reporter antigen. In parallel, we are developing techniques for assessing in a quantitative manner subtle changes in the activity of PI3K/PTEN downstream effectors, namely Akt protein kinases. We are using a new capillary isoelectric focusing (IEF) technology that allows detailed phospho-isoform profiling and relative quantification of individual phosphorylation states of a protein utilizing a single pan-specific antibody in a single run. By this approach, we have been able to achieve reliable assessment of small changes in Akt activation levels following PI3K inhibition or PTEN overexpression which was not feasible using a traditional western blotting technique.
Speaker: G. Leondaritis
Laboratory of Pharmacology, Medical School, University of Ioannina and
MRC Centre for Developmental Neurobiology, King's College London
Time: Thursday, 30 May 2013, 11:00