The PTEN phosphatase represents a key node in protein-protein interaction networks and, through binding and modulation of other signaling components, regulates diverse aspects of cellular morphology and homeostasis1. PTEN functions predominately at the cell membrane, converting phosphatidylinositol 3,4,5-trisphosphate (PIP3) to phosphatidylinositol 4,5-bisphosphate (PI4,5P2), thereby directly antagonizing the activity of phosphoinositide 3-kinase (PI3K) and its established downstream signaling pathways. Much progress has been made in characterizing the function of PTEN and its binding partners in neurons, especially during the processes of neurite outgrowth and synapse formation2-4. However, how such PTEN-based scaffolds are dynamically regulated and involved in regulating PI3K or other signaling systems is less clear.
We propose, here, to explore the role of membranous PTEN scaffolds in the vicinity of the plasma membrane, which regulate cortical actin dynamics and/or membrane trafficking during neuronal circuit formation. Our previous proteomics screen of components of PTEN-based scaffolds in neurons identified several cytosolic PTEN-interacting proteins (for example members of the Myosin V family of motorproteins and the actin-binding protein Drebrin (DBN)5, 6), as well as membrane associated proteins (for example members of a family of transmembrane Lipid Phosphatases).
To analyse the function of PTEN scaffolds we will use neurons derived directly from embryonic stem cells (ES cells) as our primary experimental system7. ES cell-derived neurons allow us to rapidly introduce genetic modifications such as fluorescent fusion proteins, cell compartment-specific variants, null mutations, and RNAi knock-downs and then to systematically assay the interaction of PTEN pathway components through epistasis experiments.
Finally, we will apply SILAC-based quantitative proteomic technologies in ES cells derived neurons in order to probe for the existence of other distinct neuronal PTEN scaffolds that associate in specific cellular compartments.
Song, M.S., Salmena, L. & Pandolfi, P.P. The functions and regulation of the PTEN tumour suppressor. Nat Rev Mol Cell Biol 13, 283-296 (2012).
Cosker, K.E. & Eickholt, B.J. Phosphoinositide 3-kinase signalling events controlling axonal morphogenesis. Biochem Soc Trans 35, 207-210 (2007).
van Diepen, M.T. & Eickholt, B.J. Function of PTEN during the formation and maintenance of neuronal circuits in the brain. Dev Neurosci 30, 59-64 (2008).
Waite, K. & Eickholt, B.J. The Neurodevelopmental Implications of PI3K Signaling. Curr Top Microbiol Immunol (2010).
Kreis, P. et al. Phosphorylation of the Actin Binding Protein Drebrin at S647 Is Regulated by Neuronal Activity and PTEN. PLoS One 8, e71957 (2013).
van Diepen, M.T. et al. MyosinV controls PTEN function and neuronal cell size. Nat Cell Biol 11, 1191-1196 (2009).
Wichterle, H., Lieberam, I., Porter, J.A. & Jessell, T.M. Directed differentiation of embryonic stem cells into motor neurons. Cell 110, 385-397 (2002).