Mf. Olson et al., MOLECULAR-BASIS FOR THE 3',5'-CYCLIC ADENOSINE-MONOPHOSPHATE RESISTANCE OF KIN MUTANT Y1 ADRENOCORTICAL TUMOR-CELLS, Molecular endocrinology, 7(4), 1993, pp. 477-487
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.