Neurological diseases and psychiatric illnesses affect more than 25% of the US population and annual medical costs are estimated at an excess of $558 billion. Genetic susceptibility to these disabilities has been noted for decades; however, many of these disorders have contributions from multiple factors including genetics, epigenetics, and the environment. In order to continue the development of our understanding of these diseases, we must understand the basic biology of the neurological system. Little is known about regulation of neuronal circuits and even less is known about the biology of the axon initial segment. The axon initial segment is a critical domain for the integration of 1000s of neurons and the most likely origin of action potentials. Recently, 480-kDa Ankyrin-G was established as the master organizer of this domain. This protein is formed by the inclusion of an alternatively spliced giant neuron-specific 7.6kb exon which is also known to be the site of many mutations associated with various neurological and psychiatric diseases including intellectual disability, bipolar disorder, schizophrenia. Using novel techniques,480-kDa Ankyrin-G has been shown to be phosphorylated at high levels in the neuronal-specific 7.6kb region. S2417 is one of these sites of high phosphorylation, and is critical for recruiting its known binding partner beta-4 spectrin to the axon initial segment. The overall goal of this proposal is to investigate the role f phosphoregulation of S2417 in regulating the axon initial segment. Having demonstrated that S2417 is phosphorylated in vivo, Specific Aim 1 tests the hypothesis that S2417 phosphorylation is regulated. First, Aim1 examines the effects of development and neuronal activity on phosphorylation in the forebrain using novel transgenic mice that expresses GFP tagged Ankyrin-G polypeptides under the control of Cre-recombinase as well as the subcellular localization of phosphorylated 480-kDa Ankyrin-G using a phospho-specific antibody against S2417. Second, Aim 1 examines the contribution of Casein kinase 2 to the phosphorylation of S2417 using genetic manipulation of the kinase in cultured neurons. Next, Aim 2 tests the hypothesis that phosphorylation of S2417 changes the physical properties of the axon initial segment by investigating intramolecular changes to 480-kDa Ankyrin-G by measuring biophysical properties. Finally, Aim 2 examines downstream consequences from loss of beta-4 spectrin at the axon initial segment using neuronal cell culture assays. This project is innovative in that it 1) is the first study of molecular regulation of the axon initial segment by focusing on 480-kDa Ankyrin-G and 2) this study utilizes novel techniques and reagents. This training plan contributes to my long-term objective of being an independent scientist in an academic research setting.