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NIDA Proteomics Center
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Investigators
> Paul Lombroso
Involvement of the Tyrosine
Phosphatase, STEP, in the Actions of Amphetamine
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. |
