Paul Lombroso, Child Study Center, Yale University
The Lombroso lab is characterizing the structure and function of the protein tyrosine phosphatase, STEP that is specifically enriched in the brain. Three substrates of STEP have been identified. These include the mitogen-activated protein kinase (MAPK) family members ERK1/2, the tyrosine kinase Fyn, and the NR2B subunit of the NMDA receptor. STEP dephosphorylates these substrates, and in the case of ERK1/2 and Fyn, inactivates them, while in the case of NR2B, STEP dephosphorylation leads to the endocytosis of the NMDA receptor complex. These results suggest that STEP may function to oppose the development of synaptic plasticity.
We have generated the STEP knockout mouse. Using the STEP knock-out mouse, we will conduct two experiments that will identify novel targets for STEP: 1. Striatal, cortical or hippocampal tissue that contain synaptic targets for STEP will be prepared from WT and STEP knock-out mice, and fractionated by differential centrifugation. Phosphotyrosine-containing proteins will be enriched from the different fractions using immobilized phosphotyrosine antibody. Purified phospho-proteins will be separated by SDS-PAGE. Following in-gel trypsin digestion, and phosphopeptide enrichment by TiO2, nLC-MS/MS (nano-Liquid Chromatography-Tandem Mass Spectrometry), analysis will be performed with a LTQ-Orbitrap mass spectrometer. The resulting high mass accuracy orbitrap generated precursor and linear ion trap generated fragment ions will be matched against the theoretical fragmentation patterns in protein databases, and will be interpreted further to determine the sites of phosphorylation on identified phosphopeptides. Comparison of the levels of phosphorylated proteins in extracts from wild-type and STEP KO mice will be carried out using either label-free methods, or following labeling of phosphopeptide pools with iTRAQ reagents. In a second study will use similar techniques to analyze tissue obtained from STEP knock-out mice given intermittent amphetamine injections and these tissues will be compared with tissues obtained from similarly treated wild type mice.