Protein phosphorylation systems of the brain are extraordinarily more active then those of other tissues and appear to represent a key brian mechanism, effecting a multitude of diverse neuronal functions. However, in contrast to the especially prominent role protein phosphorylation appears to play in brain function, very little is known regarding the genes encoding brain protein kinases, and even less is known about the true physiological role(s) such kinase fulfill in the brain. Therefore the first major objective of this proposal is to obtain fundamental information regarding the genes encoding brain protein kinases in two specific ways: 1) A detailed molecular characterization of the gene encoding a new brain Ca++/calmodulin-dependent protein kinase (CaM kinase IV) will be developed using a partial-length cDNA as a starting point. Emphasis will be placed on obtaining basic information with regard to the sequence, structural organization, expression, and regulation of this gene. We have recently determined that the human CaM kinase IV gene maps to the same chromosome position (5q22) as the gene for familial polyposis coli (FPC), and inherited form of colon cancer. Therefore, the possibility that the genes are closely linked will be investigated by a) identification of RFLPs associated with the CaM kinase IV gene and b) use os such RFLPs in linkage analyses with FPC families. 2) A novel molecular cloning strategy, homology probing will be utilized to identify genes encoding previously uncharacterized protein-serine kinases present in the brain. In this approach brain cDNA libraries will be screened with two sets of degenerate oligonucleotide probes derived from conserved regions of the catalytic domain of known protein-serine kinase. Once a clone has been identified by sequence as encoding a new serine kinase, a detailed characterization of the corresponding gene, in the manner described above for the CaM kinase IV gene, will be initiated, with priority given to those kinases that appear to be brain-specific. In addition, a second approach, i.e. he use of 125I-calmodulin as a probe to screen brain expression libraries, will also be explored. The second major objective of this proposal is to obtain information about the true physiological function(s) of these new kinases in the brain. Initial studies will utilize synthetic peptides, derived from the deduced amino acid sequence of the cloned kinase cDNAs, to generate antibodies of predetermined specificity. Such antibodies would be used to isolate the corresponding kinase which, in turn, would then be subjected to biochemical and functional characterization, including, for example, analysis of catalytic activity, second-messenger dependence, and substrate specificity. The antibodies would also be used in immunocytochemical analyses of brain at the light and electron microscopic levels to obtain information about the regional, cellular, and subcellular distribution of the kinase. As a longterm strategy, the use of transgenic nice will be explored as a possible approach to obtaining a more comprehensive understanding of the in vivo function of these kinases. While technically challenging, the ability to produce, and then analyze at multiple levels, mice in which specific kinase genes are under- or over-expressed would represent a powerful approach to understanding the biological role(s) such proteins fulfill in the brain.
