Calcium is a fundamental constituent of life and is critical to the normal functioning and equilibrium of multicellular organisms. As part of this homeostasis, transient increases in the intracellular concentration of free calcium (calcium signals) are generated in response to a variety of specific and diffuse stimuli (e.g., hormone or drug action via classical cell surface or intracellular receptors as well as stretch and gravity through less well defined mechanisms). Regardless of the source of cell stimulus or calcium release, the most well-established pathways for transducing an intracellular calcium signal into a biological response is through its detection, decoding and translation by a specific class of proteins for which calmodulin (CaM) is a prototype. CaM is present in all eukaryotic cells and is the calcium regulatory subunit of a variety of enzymes, cytoskeletal structures, and membrane transport complexes. A long-term goal of our research is to understand how a eukaryotic cell calcium signal is transduced into a biological response through CaM mediated pathways, especially protein kinases. The short-term goals of this research are to increase our knowledge about the encoding, assembly, targeting, and calcium signal transduction mechanism of a prototype CaM:enzyme complex in order to gain insight into fundamental principles and molecular themes used by the CaM pathways. The specific scientific hypotheses being addressed in the proposed investigations are: 1. A single genetic locus in vertebrates encodes a set of CaM regulated proteins involved in the regulation of cell structure and function through their interactions with a myosin II molecular motor system; 2. The physiologically important target for intracellular calcium signals is the CaM molecule in the context of a supramolecular structure which, in normal cell function, can enhance the calcium sensitivity of CaM or, in potential pathological situations, can suppress the phenotypic effects of deleterious mutations in CaM; 3. A new class of inhibitors can be developed through the use of our knowledge of structure, function and mechanism. Specifically, it will be determined if the novel genetic locus that encodes nonmuscle/smooth muscle myosin light chain kinase (nm/smMLCK) and the kinase related protein (KRP), two proteins involved in the regulation of cell structure and function through their interactions with myosin II, encode an additional protein that is potentially a new CaM binding regulatory protein. In addition, the experimentally tractable CaM recognition peptide that is common for MLCK and this new CaM binding protein will be used to determine the basis of the functional link between selective calcium recognition and enzyme recognition by CaM. These studies are logical extensions of the results obtained during our previous funding period and have the opportunity to provide knowledge about fundamental aspects of normal cell functioning, and may provide insight into disease processes and suggest approaches to the development of new classes of therapeutics.
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