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.