The main focus of the project is calcium/calmodulin-dependent protein kinase II (CaMKII), a major protein in the postsynaptic density (PSD) and a calcium-regulated enzyme, implicated in long-term synaptic modification and memory. Both structural and enzymatic roles of CaMKII are considered as putative mechanisms in mediating calcium-induced modifications in the PSD. The structure of isolated soluble CaMKII has been studied by electron microscopy. Replicas obtained by rapid freezing and rotary shadowing of the molecules adsorbed on mica reveal a rosette-shaped oligomeric structure with 8-10 peripheral particles arranged around a central association domain. Mutant CaMKII molecules have been generated to explore the contribution of selected peptide sequences to the overall structure of the holoenzyme. One of these projects has recently been completed and provides evidence that, contrary to the general belief, the sequence of amino acids 317-328 does not constitute the thin link between the central assembly region and the peripheral catalytic region of the rosette shaped molecule. In order to observe the organization of the CaMKII molecule within the PSD, the structure was chemically dissected with various reagents. The best strategy appears to be treatment with urea which removes actin, tubulin and spectrin to yield an insoluble fraction highly enriched in CaMKII. CaMKII autophosphorylates on multiple sites in the absence and presence of calcium, with distinct functional consequences. This property of the enzyme could allow the detection of the temporal pattern of postsynaptic calcium transients and thus provide a mechanism for synaptic frequency detection. Possible modification in the autophosphorylation properties of CaMKII as a result of its translocation to the PSD is explored. The threshold levels of calcium-dependent phosphorylation necessary to trigger calcium-independent phosphorylation for cytosolic and PSD- associated kinases are compared and autophosphorylation sites on soluble and PSD-associated enzymes are being identified by mass spectrometry. In order to develop a system to test the predictions of the model proposed, preliminary studies on the autophosphorylation of CaMKII in hippocampal cell cultures are conducted in collaboration with Dr. Ralston's group. In a new initiative with Dr. Vinade, who recently joined the laboratory, regulation of the phosphorylation of PSD- associated AMPA type glutamate receptor (GluR1) is being explored. Phosphorylation of the receptor by the calcium-activated and autonomous forms of CaMKII and the identity of the phosphatases responsible for its dephosphorylation are studied.
