Structure-function relationships in regulatory subunit (R) of cyclic AMP-dependent protein kinase will be studied using R structural mutations in cultured S49 mouse lymphoma cells. Wild-type S49 cells are growth-inhibited and cytolyzed by exposure to cyclic AMP (cAMP) or its analogs; cAMP-resistance can be conferred by R mutations that increase the concentrations of cAMP required for kinase activation. Mutants with lesions of this sort will be collected in both diploid S49 cells and in S49 sublines hemizygous for R gene expression. Cyclic AMP-sensitive revertants of resistant mutants will also be isolated to obtain """"""""second-site"""""""" mutations affecting R function. A variety of cellular and biochemical assays will be employed to characterize lesions affecting particular R functions, such as--cyclic nucleotide binding to the two cAMP-binding sites, binding and inhibition of catalytic subunit, and cooperative interactions between cAMP-binding sites and between cAMP-binding and catalytic subunit-binding sites. Mutations affecting R charge will be identified by two-dimensional gel electrophoresis of affinity-purified, radiolabeled R. Such """"""""charge-shift"""""""" mutations will be localized within the R protein by a novel peptide mapping procedure that employs two-dimensional gel electrophoresis of overlapping R fragments generated by partial proteolysis of the denatured protein. This procedure, which has been elaborated in preliminary studies, will be refined to allow mapping of all charge-shift mutations to 5% or less of the R polypeptide chain. From selected mutant sublines, R genes will be cloned to allow DNA sequence comparisons between wild-type and mutant genes. Studies constituting this project should provide new insights into the molecular regulation of cAMP-dependent protein kinase, an enzyme essential for regulating cellular growth, differentiation, and hormonal responses. Kinase mutations have been implicated in the progression of human tumors. Furthermore, mapping procedures to be refined under this project will fill a conspicuous gap that has prevented widespread application of somatic cell genetics to problems of mammalian protein structure.
