(from abstract) The long-term goal of the proposed studies is to determine the role of Ca2+/ calmodulin-dependent protein kinase II (CaM-kinase II) in regulating neuronal functions and specifically, synaptic functions. Alterations in synaptic function affect learning and memory and aberrant synaptic function is implicated in many neuropsychiatric and neuropathologic disorders. CaM-kinase II is the most abundant kinase in the brain and is highly concentrated at synapses, however, different isoforms of CaM-kinase II are found distributed through most tissues in the body and its functions in cellular regulation are diverse. The complex isolated from the forebrain is composed of multiple copies of two main subunits, termed a and b. a is considered a neuron-specific isoform. Other isoforms, g and d, are also present in the brain and are the main isoforms found outside the nervous system. Although the catalytic and regulatory domains of all four isoforms have homologous amino acid sequences, significant differences exist in their C-terminal domains. This proposal will determine the significance of certain domains and amino acid residues on the enzymatic function, association, structural architecture, and subcellular localization of the subunits. Through protein engineering, CaM-kinase II variants were constructed to map the association, regulatory and catalytic domains in the amino acid sequence. The map of the association domain will be further refined using biophysical techniques to delineate residues that are related to the formation of holoenzymes. 3-D electron microscopy will be employed to determine the architecture of some of these variants and the structure of the g and d isoforms. Structure-function relationships will be established for these complexes. The 3-D structure of Fab-labeled a and b complexes will determine the locations and disposition of the C-terminal domains in these structures. The determination of the 3-D structure of the activated a complex will assess the impact of its activation n the architecture of its scaffold and arm-like features identified in our non-activated structure. Confocal and live cell fluorescence imaging will determine the location of tagged isoforms of CaM-kinase II in neurons and determine the consequence of cell signaling on their distribution. Protein engineering will expand these studies to investigate the significance of amino acid residues in targeting the enzyme to intracellular structures.
Showing the most recent 10 out of 31 publications
