Calcium/calmodulin-dependent protein kinases (CaM-kinases) are important signal transduction enzymes which are particularly abundant in brain. These kinases are crucial for regulating numerous Ca2+-triggered physiological functions including hormone actions, neurotransmitter synthesis and release, modulating several ion channels, mediating aspects of learning and memory, and triggering selective transcription of key genes. This proposal will examine important structural and regulatory properties of three CaM-kinases: CaM-kinase II, CaM-kinase IV and CaM-kinase kinase. These studies will utilize purified enzymes in vitro as well as studies on mammalian cells transfected with wild- type and mutants of these kinases. Techniques of protein chemistry, site-specific mutagenesis and enzymology will be employed. Major components of these studies will include: 1. Cloning and characterization of a protein inhibitor of CaM-KII. The biochemical mechanism(s) regulating its interaction with and inhibition of CaM-KII will be determined in vitro and in cultured cells. 2. We will study mechanisms (e.g., nuclear localization signals) involved in regulating the distribution of CaM-KIV between the nucleus, where it regulates gene transcription, and the cytosol, where it exerts cross-talk with the MAP-kinase pathways. We will also determine the biochemical mechanism by which Ca2+independent activity is generated in CaM-kinase IV upon its activation by CaM-kinase kinase. 3. Extensive studies will be performed on structure/function aspects of the a and b isoforms of CaM-kinase kinase. In particular, regulatory functions of unique sequences in these proteins will be examined in terms of their potential autoregulatory properties or interactions with other cellular proteins. Activation by hormones and neurotransmitters will be assessed. These studies will further our understanding of the enzymatic and regulatory properties of these key signaling proteins, thereby furthering our understanding of their roles in hormone actions, learning and memory, epilepsy and stroke.
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