CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE II

The objective of these studies is to delineate the molecular
mechanisms by which brain Ca++/calmodulin(Cam)-dependent protein
kinase II (CaM-kinase II) is regulated by binding of Ca /CaM and
by autophosphorylation, both with the purified kinase and in intact
cells and tissues. Long-term regulation of brain CaM-kinase II by
covalent mechanisms (i.e., autophosphorylation) is of special
interest because of the putative neuronal functions of this kinase.
There is good evidence that presynaptically CaM-kinase II is
intimately involved in Ca-dependent regulation of catecholamine
biosynthesis and neurotransmitter exocytosis. CaM-kinase II
comprises 50% of protein in the postsynaptic density of excitatory
synapses and may be involved in long-term alterations of synaptic
plasticity. Previous studies from this and other laboratories have led to the
formulation of a model for the regulation of CaM-kinase II by
binding of Ca/CaM and by autophosphorylation. This model will be
tested using synthetic peptides, derived from the known sequence
of the 50 kDa subunit of the brain CaM-kinase II, to characterize
CaM-binding and substrate-directed inhibitory domains within the
kinase sequence. Amino acids essential to these functions will be
identified using altered peptide sequences. The effects of
phosphorylation of consensus phosphorylation sites flanking these
two domains will be assessed in terms of the function of these
domains. These results will be utilized in the design of in vitro
site-specific mutagenesis to test the functions of specific amino
acids on the properties of the kinase. Lastly the ability of CaM-
kinase II to undergo autophosphorylation and formation of Ca
independent species in intact cells will be assessed in hippocampal
brain slices and in cultured hippocampal cells and PC12 cells in
response to Ca influx. These studies will further our
understanding of the regulation and function of this
multifunctional Ca++ -dependent kinase.