The proposed studies will examine the structure and function of a major protein kinase in vertebrate neural tissues designated Ca2+/calmodulin-dependent protein kinase II (CaM-KII). CaM-KII constitutes a major enzymatic component of asymmetric synaptic contacts and comprises most of the protein mass of postsynaptic densities (PSDs). Synapses are the sites of cell-cell communication in brain and have been the focus of studies on neuronal plasticity associated with learning and memory, synaptic remodeling following lesion and CNS development. Indeed, abnormalities that are likely to compromise synaptic efficacy may contribute to a variety of neurological disorders. Because of its abundance, regulatory properties and key subcellular localization at neuronal PSDs, CaM-KII is thought to play a pivotal role in calcium-dependent events underlying synaptic transmission. CaM-KII is composed of two distinct protein subunits (50 and 60 kDa); both bind calmodulin and are assembled to make the large holoenzyme complex. CaM-KII possesses an important regulatory property in that the activation-dependent phosphorylation (i.e., """"""""autophosphorylation"""""""") converts the kinase into a constitutive enzyme that no longer requires Ca2+/calmodulin for activity. Thus, CaM-KII has been proposed to constitute a reversible molecular """"""""switch"""""""" that transduces intracellular Ca2+ signals at synapses into protein phosphorylation events that underlie cell-cell communication and synaptic plasticity. The genes for both CaM-KII subunits have recently been cloned and sequenced. We have used recombinant DNA techniques and biochemistry to identify the regulatory calmodulin-binding domain of the 50 kDa subunit of CaM-KII. Preliminary results have identified the putative autophosphorylation sites of this subunit that may be responsible for converting the highly regulated CaM- KII into the Ca2+/calmodulin-independent enzyme. We will utilize genetic engineering to elucidate functional interactions between the calmodulin-binding and autophosphorylation domains of CaM-KII. Recombinant DNA strategies will be used to elucidate structure-function relationships and interactions between the two subunits that comprise the CaM-KII holoenzyme. We will examine at both nucleic acid and polypeptide levels the expression and assembly of the CaM-KII holoenzyme in vitro, during neuronal differentiation. The accumulation of CaM-KII at synapses is developmentally regulate moveover, the genes for its two subunits are differentially expressed during development. We propose studies to understand how local environmental signals (e.g., trophic factors and neuromodulators) effect the expression of CaM-KII genes and to detail intracellular events following gene transcription. The long-term significance of these studies lies in their ability to answer important questions about the structure and function of CaM-KII, and the role that this enzyme plays in neuronal protein phosphorylation, synapse formation, cell-cell communication and neuronal plasticity in the mammalian CNS.
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