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NIDA Proteomics Center
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Investigators
> Susumu Tomita
Identify Calcium Regulated
Phosphorylation Sites In Stargazin-Like TARPs
Susumu Tomita, Department of Molecular Biophysics and Biochemistry,
Yale University Neuronal
circuits store information in the brain. Billions of neurons in the brain
communicate with each other at synapses. Synaptic strength in neuronal circuits
is regulated by neuronal activity through several mechanisms including protein
phosphorylation. Dysregulation of synaptic transmission causes several
neurological diseases including epilepsy, autism, mental retardation and
neurodegeneration by alterations in neural circuits. Indeed, glutamatergic
antagonists, which are non-competitive inhibitors of NMDA type glutamate
receptor, phencyclidine (PCP) and ketamine, can produce both positive and
negative symptoms associated with schizophrenia in healthy human, and worsen
these symptoms in schizophrenia patients. An important function of NMDA
receptors is to regulate protein phosphorylation cascades through calcium influx
into neurons, thus it is likely that non-competitive NMDAR inhibitors effect
these protein phosphorylation pathways. It has been also shown that calcium
dependent protein kinases (PKC and CaMKII) are regulated by NMDA receptors to
control synaptic strength. However, the lack of robust and specific inhibitors
of kinases makes it difficult to identify kinase substrates downstream of
synaptic activation. Our search for kinase substrates focuses on AMPA receptor
interacting proteins since AMPA receptor are the molecules regulated by NMDA-dependent
neuronal activity to control synaptic strength. AMPA receptors have as auxiliary
subunits, TARP. TARP phosphorylation has been shown to play critical roles in
controlling synaptic strength. However, it remains unclear how TARP
phosphorylation is regulated to modulate synaptic AMPA receptors. To understand
mechanisms of AMPA receptor regulation, we need to identify the sites in TARP
phosphorylated by PKC and CaMKII downstream of NMDA receptors upon neuronal
activation. Here, we will identify sites in TARP cytoplasmic domain
phosphorylated by PKC and CaMKII in vitro by combination of radio-labeled
proteins and the mass spectrometry analysis using MALDI-TOF-TOF. Furthermore, we
will evaluate effects of neuronal stimulation including multiple drugs on
phosphorylation of TARP in vivo by multiple proteomic approaches. These
proteomic analyses will reveal critical phosphorylated substrates regulated by
synaptic transmission and will identify new drug targets for neurological
diseases. |
