MOLECULAR-BASIS FOR THE 3',5'-CYCLIC ADENOSINE-MONOPHOSPHATE RESISTANCE OF KIN MUTANT Y1 ADRENOCORTICAL TUMOR-CELLS

A series of mutant cell lines (Kin) were previously isolated from Y1 a drenocortical tumor cells based on their ability to resist the growth- inhibitory effects of 8-bromo cAMP. In these Kin clones, cAMP-dependen t protein kinase (cAMPdPK) was resistant to activation by cAMP as the consequence of mutations affecting the type I regulatory subunit (RI) of the enzyme. This study shows that the cAMP-resistant phenotypes of mutant clones Kin-2, Kin-7, and Kin-8 were associated with single base changes causing substitutions, respectively, of Glu for Gly200, Trp f or Arg334, and Asp for Gly324 in the RI protein. By expressing the mut ant Trp334 and Asp324 forms of RI under the control of an inducible pr omoter in Y1 cells, the causal relationship between these RI mutations and impairment of cAMP-stimulated adrenocortical responses was studie d. Expression of the mutant RI forms rendered cAMPdPK resistant to act ivation by cAMP and decreased cAMP-stimulated cell rounding, steroid p roduction, and growth inhibition. These observations indicate that the cAMP-resistant phenotype of Kin mutant clones resulted specifically f rom single mutational events in RI and thus establish the importance o f cAMPdPK as an essential regulator of adrenocortical function. Unlike the original Kin mutant clones, transformants expressing the mutant f orms of RI had adenylyl cyclases that were resistant to activation by ACTH, forskolin, or sodium fluoride. These results indicate that there may be a hitherto unappreciated mechanism of regulation of adenylyl c yclase activity by cAMPdPK.